Compositions And Methods Comprising Variant Microbial Proteases

ABSTRACT

The present invention provides variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions. In some embodiments, the present invention provides variant subtilisins suitable for laundry cleaning applications.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 12/996,564, filed Jun. 14, 2011, which is a U.S. National StageApplication of International Application No. PCT/US2009/046066, filedJun. 3, 2009, which claims the benefit of U.S. Provisional ApplicationNo. 61/059,695, filed Jun. 6, 2008, which are herein incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention provides variant subtilisins and compositionscomprising at least one variant subtilisin set forth herein, as well asmethods for using these variants and compositions. In particular, thepresent invention provides variant proteases suitable for laundrycleaning applications.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolases comprising adiverse class of enzymes having a wide range of specificities andbiological functions. Much research has been conducted on thesubtilisins, due largely to their usefulness in cleaning and feedapplications. Additional work has been focused on the adverseenvironmental conditions (e.g., exposure to oxidative agents, chelatingagents, extremes of temperature and/or pH) which can adversely impactthe functionality of these enzymes in various applications. Nonetheless,there remains a need in the art for enzyme systems that are able toresist these adverse conditions and retain or have improved activityover those currently known in the art.

SUMMARY OF THE INVENTION

The present invention provides variant subtilisins and compositionscomprising at least one variant subtilisin set forth herein, as well asmethods for using these variants and compositions. In some embodiments,the present invention provides variant proteases suitable for laundrycleaning applications.

The present invention provides isolated subtilisin variants comprisingat least one set of the following substitution sets: G97A-G128A-Y217Q,G97A-L126A-G128A, G97A-L126A-G128A-Y217Q, G97A-L126A-Y217Q,G97A-M124V-G128A, G97A-M124V-G128A-Y217Q, G97A-M124V-L126A,G97A-M124V-L126A-G128A, G97A-M124V-L126A-Y217Q, G97A-M124V-Y217Q,G97A-N123G-G128A, G97A-N123G-G128A-Y217Q, G97A-N123G-L126A,G97A-N123G-L126A-G128A, G97A-N123G-L126A-Y217Q, G97A-N123G-M124V,G97A-N123G-M124V-G128A, G97A-N123G-M124V-L126A, G97A-N123G-M124V-Y217Q,G97A-N123G-Y217Q, L126A-G128A-Y217Q, L96T-G128A-Y217Q, L96T-G97A-G128A,L96T-G97A-G128A-Y217Q, L96T-G97A-L126A, L96T-G97A-L126A-G128A,L96T-G97A-L126A-Y217Q, L96T-G97A-M124V, L96T-G97A-M124V-G128A,L96T-G97A-M124V-L126A, L96T-G97A-M124V-Y217Q, L96T-G97A-N123G,L96T-G97A-N123G-G128A, L96T-G97A-N123G-L126A, L96T-G97A-N123G-M124V,L96T-G97A-N123G-Y217Q, L96T-G97A-Y217Q, L96T-L126A-G128A,L96T-L126A-G128A-Y217Q, L96T-L126A-Y217Q, L96T-M124V-G128A,L96T-M124V-G128A-Y217Q, L96T-M124V-L126A, L96T-M124V-L126A-G128A,L96T-M124V-L126A-Y217Q, L96T-M124V-Y217Q, L96T-N123G-G128A,L96T-N123G-G128A-Y217Q, L96T-N123G-L126A, L96T-N123G-L126A-G128A,L96T-N123G-L126A-Y217Q, L96T-N123G-M124V, L96T-N123G-M124V-G128A,L96T-N123G-M124V-L126A, L96T-N123G-M124V-Y217Q, L96T-N123G-Y217Q,M124V-G128A-Y217Q, M124V-L126A-G128A, M124V-L126A-G128A-Y217Q,M124V-L126A-Y217Q, N123G-G128A-Y217Q, N123G-L126A-G128A,N123G-L126A-G128A-Y217Q, N123G-L126A-Y217Q, N123G-M124V-G128A,N123G-M124V-G128A-Y217Q, N123G-M124V-L126A, N123G-M124V-L126A-G128A,N123G-M124V-L126A-Y217Q, N123G-M124V-Y217Q, N62Q-G128A-Y217Q,N62Q-G97A-G128A, N62Q-G97A-G128A-Y217Q, N62Q-G97A-L126A,N62Q-G97A-L126A-G128A, N62Q-G97A-L126A-Y217Q, N62Q-G97A-M124V,N62Q-G97A-M124V-G128A, N62Q-G97A-M124V-L126A, N62Q-G97A-M124V-Y217Q,N62Q-G97A-N123G, N62Q-G97A-N123G-G128A, N62Q-G97A-N123G-L126A,N62Q-G97A-N123G-M124V, N62Q-G97A-N123G-Y217Q, N62Q-G97A-Y217Q,N62Q-L126A-G128A, N62Q-L126A-G128A-Y217Q, N62Q-L126A-Y217Q,N62Q-L96T-G128A, N62Q-L96T-G128A-Y217Q, N62Q-L96T-G97A,N62Q-L96T-G97A-G128A, N62Q-L96T-G97A-L126A, N62Q-L96T-G97A-M124V,N62Q-L96T-G97A-N123G, N62Q-L96T-G97A-Y217Q, N62Q-L96T-L126A,N62Q-L96T-L126A-G128A, N62Q-L96T-L126A-Y217Q, N62Q-L96T-M124V,N62Q-L96T-M124V-G128A, N62Q-L96T-M124V-L126A, N62Q-L96T-M124V-Y217Q,N62Q-L96T-N123G, N62Q-L96T-N123G-G128A, N62Q-L96T-N123G-L126A,N62Q-L96T-N123G-M124V, N62Q-L96T-N123G-Y217Q, N62Q-L96T-Y217Q,N62Q-M124V-G128A, N62Q-M124V-G128A-Y217Q, N62Q-M124V-L126A,N62Q-M124V-L126A-G128A, N62Q-M124V-L126A-Y217Q, N62Q-M124V-Y217Q,N62Q-N123G-G128A, N62Q-N123G-G128A-Y217Q, N62Q-N123G-L126A,N62Q-N123G-L126A-G128A, N62Q-N123G-L126A-Y217Q, N62Q-N123G-M124V,N62Q-N123G-M124V-G128A, N62Q-N123G-M124V-L126A, N62Q-N123G-M124V-Y217Q,and N62Q-N123G-Y217Q, wherein the substitutions are at positionsequivalent to the positions of BPN′ subtilisin set forth in SEQ ID NO:1.

The present invention also provides isolated subtilisin variantscomprising at least one set of the following substitution sets:G97N-G128A-Y217M, G97G-G128S-Y217E, G97A-G128A-Y217Q, G97M-G128S-Y217E,G97A-G128S-Y217Q, G97D-G128S-Y217Q, G97M-G128G-Y217M, G97G-G128S-Y217Q,G97S-G128S-Y217Q, G97G-G128A-Y217Q, G97S-G128A-Y217E, G97A-G128S-Y217L,G97A-G128A-Y217N, G97Q-G128S-Y217L, G97A-G128A-Y217M, G97A-G128A-Y217S,G97D-G128A-Y217Q, G97M-G128S-Y217Q, G97Q-G128G-Y217D-S87Y,G97S-G128A-Y217N, G97A-G128S-Y217T, G97D-G128S-Y217E, G97D-G128A-Y217L,G97G-G128S-Y217E-S78P-A272T, G97T-G128S-Y217D, G97D-G128A-Y217I,G97Q-G128S-Y217Q, G97G-G128A-Y217D, G97Q-G128A-Y217N, G97S-G128A-Y217M,G97S-G128S-Y217N, G97S-G128S-Y217M, G97E-G128S-Y217M, G97S-G128P-Y217Q,G97T-G128S-Y217Q, G97D-G128S-Y217Q-A73T, G97E-G128S-Y217N,G97G-G128A-Y217I, G97Q-G128A-Y217D, G97Q-G128S-Y217M,G97R-G128T-Y217Q-S162P, G97S-G128S-Y217D, G97T-G128P-Y217I,G97Q-G128G-Y217E, G97C-G128G-Y217N, G97D-G128S-Y217H, G97M-G128S-Y217L,G97M-G128S-Y217N, G97S-G128S-Y217E, G97M-G128S-Y217I, G97A-G128P-Y217A,G97R-G128S-Y217D, G97D-G128A-Y217D, G97V-G128G-Y217D, G97V-G128G-Y217E,G97A-G128G-Y217T, G97G-G128N-Y217L, G97D-G128A-Y217T, G97M-G128A-Y217E,and G97M-G128A-Y217N, wherein the substitutions are at positionsequivalent to the positions of BPN′ subtilisin set forth in SEQ ID NO:1.

The present invention further provides isolated subtilisin variantscomprising at least one set of the following substitution sets:S24R-S87D-Q206E, P40E-A144K-K213L, N61E-P129E-S159K, S87D-S162K-K265N,S87D-T242R-Q275E, N61E-Q103E-N240K, S87D-T242R-K265N, N62R-K265N-Q275E,P129E-S145D-N240K, P129E-P239R-K265N, Q103E-P129E-T242R,P40E-N61E-S87D-S162K-T242R, S24R-N62R-S87D-S145D-K265N,P40E-N62R-S87D-Q103E-S162K, S24R-P40E-S145D-S159K-K213L,S24R-S87D-A144K-K265N-Q275E, N61E-P129E-S162K-K213L-N240K,N61E-S145D-S162K-K213L-T242R, S87D-A144K-S145D-S159K-Q275E,S24R-P129E-Q206E-N240K-K265N, N61E-Q103E-A144K-K213L-T242R,N62R-S159K-Q206E-K265N-Q275E, S24R-Q103E-P129E-N240K-K265N,N61E-Q103E-P129E-P239R-N240K, P129E-S145D-N240K-T242R-K265N,Q103E-S162K-Q206E-K213L-P239R, P40E-N61E-N62R-S87D-S159K-S162K-K265N,S24R-P40E-N61E-A144K-Q206E-K213L-T242R,P40E-N61E-S87D-P129E-S159K-S162K-T242R,P40E-N62R-S87D-S145D-S159K-S162K-Q275E,P40E-N62R-S87D-Q103E-A144K-S159K-Q275E,P40E-N62R-S87D-S159K-S162K-K265N-Q275E,P40E-N61E-P129E-A144K-S162K-K213L-N240K,P40E-N61E-Q103E-A144K-S159K-S162K-Q275E,P40E-N61E-Q103E-S159K-S162K-K213L-P239R,P40E-N61E-Q103E-S159K-S162K-K213L-N240K,N62R-S87D-P129E-S145D-S159K-S162K-Q275E,S24R-N61E-Q103E-P129E-K213L-N240K-T242R,P40E-N62R-S145D-S159K-S162K-Q206E-Q275E,N62R-S87D-S145D-S159K-S162K-K265N-Q275E,N61E-S87D-Q103E-S159K-S162K-K213L-T242R,S24R-N61E-Q103E-P129E-Q206E-P239R-N240K,S24R-N62R-P129E-S145D-P239R-K265N-Q275E,S24R-N62R-P129E-Q206E-N240K-K265N-Q275E,P40E-S145D-S159K-S162K-K213L-P239R-Q275E,N61E-Q103E-A144K-Q206E-K213L-N240K-T242R,S24R-Q103E-P129E-S145D-P239R-N240K-K265N,N61E-Q103E-P129E-K213L-P239R-N240K-T242R,N61E-Q103E-Q206E-K213L-P239R-N240K-T242R,S24R-P40E-N61E-S87D-Q103E-S159K-S162K-K213L-N240K,N61E-N62R-S87D-Q103E-S159K-S162K-K213L-T242R-Q275E,P40E-N62R-S87D-S145D-S159K-S162K-N240K-K265N-Q275E,N62R-S87D-S145D-S159K-S162K-K213L-N240K-K265N-Q275E,S24R-N61E-Q103E-P129E-Q206E-K213L-P239R-N240K-T242R,S24R-N61E-Q103E-P129E-S145D-P239R-N240K-T242R-K265N,N61E-S87D-Q103E-P129E-S159K-S162K-K213L-N240K-T242R,P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,S24R-Q103E-P129E-S145D-Q206E-P239R-N240K-T242R-K265N,N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,S24R-P40E-N61E-S87D-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,S24R-P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R-K265N,S24R-P40E-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,S24R-P40E-N61E-Q103E-P129E-A144K-S145D-K213L-P239R-N240K-T242R,S24R-N61E-S87D-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,P40E-N61E-S87D-Q103E-S145D-S159K-S162K-K213L-P239R-N240K-T242R,S24R-P40E-N61E-Q103E-P129E-S162K-Q206E-K213L-P239R-N240K-T242R,S24R-N61E-Q103E-P129E-A144K-S145D-Q206E-K213L-P239R-N240K-T242R,P40E-N61E-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R,S24R-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R-K265N, andS24R-P40E-N61E-S87D-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R,wherein said substitutions are at positions equivalent to the positionsof BPN′ subtilisin set forth in SEQ ID NO:1.

The present invention also provides subtilisin variants comprising thesubstitutions G97A/G128A/Y217Q and further comprising at least one ofthe above substitution sets, and wherein the positions correspond to thepositions of BPN′ subtilisin of SEQ ID NO:1.

The present invention further provides isolated subtilisin variantscomprising at least one set of the following substitution sets:S53G-F58G, S53G-S78N, S53G-Y104N, S53G-I111V, S53G-A114G, S53G-N117S,S53G-S125A, S53G-S132N, S53G-P239V, F58G-S78N, F58G-Y104N, F58G-I111V,F58G-A114G, F58G-N117S, F58G-S125A, F58G-S132N, F58G-P239V, S78N-Y104N,S78N-I111V, S78N-A114G, S78N-N117S, S78N-S125A, S78N-S132N, S78N-P239V,Y104N-I111V, Y104N-A114G, Y104N-N117S, Y104N-S125A, Y104N-S132N,Y104N-P239V, I111V-A114G, I111V-N117S, I111V-S125A, I111V-S132N,I111V-P239V, A114G-N117S, A114G-S125A, A114G-S132N, A114G-P239V,N117S-S125A, N117S-S132N, N117S-P239V, S125A-S132N, S125A-P239V,S132N-P239V, S53G-F58G-S78N, S53G-F58G-Y104N, S53G-F58G-I111V,S53G-F58G-A114G, S53G-F58G-N117S, S53G-F58G-S125A, S53G-F58G-S132N,S53G-F58G-P239V, S53G-S78N-Y104N, S53G-S78N-I111V, S53G-S78N-A114G,S53G-S78N-N117S, S53G-S78N-S125A, S53G-S78N-S132N, S53G-S78N-P239V,S53G-Y104N-I111V, S53G-Y104N-A114G, S53G-Y104N-N117S, S53G-Y104N-S125A,S53G-Y104N-S132N, S53G-Y104N-P239V, S53G-I111V-A114G, S53G-I111V-N117S,S53G-I111V-S125A, S53G-I111V-S132N, S53G-I111V-P239V, S53G-A114G-N117S,S53G-A114G-S125A, S53G-A114G-S132N, S53G-A114G-P239V, S53G-N117S-S125A,S53G-N117S-S132N, S53G-N117S-P239V, S53G-S125A-S132N, S53G-S125A-P239V,S53G-S132N-P239V, F58G-S78N-Y104N, F58G-S78N-I111V, F58G-S78N-A114G,F58G-S78N-N117S, F58G-S78N-S125A, F58G-S78N-S132N, F58G-S78N-P239V,F58G-Y104N-I111V, F58G-Y104N-A114G, F58G-Y104N-N117S, F58G-Y104N-S125A,F58G-Y104N-S132N, F58G-Y104N-P239V, F58G-I111V-A114G, F58G-I111V-N117S,F58G-I111V-S125A, F58G-I111V-S132N, F58G-I111V-P239V, F58G-A114G-N117S,F58G-A114G-S125A, F58G-A114G-S132N, F58G-A114G-P239V, F58G-N117S-S125A,F58G-N117S-S132N, F58G-N117S-P239V, F58G-S125A-S132N, F58G-S125A-P239V,F58G-S132N-P239V, S78N-Y104N-I111V, S78N-Y104N-A114G, S78N-Y104N-N117S,S78N-Y104N-S125A, S78N-Y104N-S132N, S78N-Y104N-P239V, S78N-I111V-A114G,S78N-I111V-N117S, S78N-I111V-S125A, S78N-I111V-S132N, S78N-I111V-P239V,S78N-A114G-N117S, S78N-A114G-S125A, S78N-A114G-S132N, S78N-A114G-P239V,S78N-N117S-S125A, S78N-N117S-S132N, S78N-N117S-P239V, S78N-S125A-S132N,S78N-S125A-P239V, S78N-S132N-P239V, Y104N-I111V-A114G,Y104N-I111V-N117S, Y104N-I111V-S125A, Y104N-I111V-S132N,Y104N-I111V-P239V, Y104N-A114G-N117S, Y104N-A114G-S125A,Y104N-A114G-S132N, Y104N-A114G-P239V, Y104N-N117S-S125A,Y104N-N117S-S132N, Y104N-N117S-P239V, Y104N-S125A-S132N,Y104N-S125A-P239V, Y104N-S132N-P239V, I111V-A114G-N117S,I111V-A114G-S125A, I111V-A114G-S132N, I111V-A114G-P239V,I111V-N117S-S125A, I111V-N117S-S132N, I111V-N117S-P239V,I111V-S125A-S132N, I111V-S125A-P239V, I111V-S132N-P239V,A114G-N117S-S125A, A114G-N117S-S132N, A114G-N117S-P239V,A114G-S125A-S132N, A114G-S125A-P239V, A114G-S132N-P239V,N117S-S125A-S132N, N117S-S125A-P239V, N117S-S132N-P239V,S125A-S132N-P239V, N76D-D120H-K213N-M222Q, wherein said substitutionsare at positions equivalent to the positions of BPN′ subtilisin setforth in SEQ ID NO:1.

The present invention also provides subtilisin variants comprising thesubstitutions G97A/G128A/Y217Q and further comprising at least one ofthe above substitution sets, and wherein the positions correspond to thepositions of BPN′ subtilisin of SEQ ID NO:1.

The present invention also provides isolated nucleic acids encoding thesubtilisin variants set forth herein, as well as expression vectorscomprising these nucleic acids, and host cells comprising theseexpression vectors.

The present invention further provides cleaning compositions comprisingat least one of the subtilisin variant provided herein. In someembodiments, the cleaning compositions are laundry detergents. In someadditional embodiments, the laundry detergent is a heavy duty liquidlaundry detergent. In some further embodiments, the cleaning compositionis a dish detergent. In some still further embodiments, the cleaningcomposition is a hard surface cleaning composition. In some additionalembodiments, the cleaning compositions further comprise one or moreadditional enzymes or enzyme derivatives selected from the groupconsisting of hemicellulases, peroxidases, proteases, cellulases,xylanases, lipases, phospholipases, esterases, cutinases, pectinases,keratinases, reductases, oxidases, phenol oxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof. In some embodiments, the cleaningcompositions further comprise at least one stabilizing agent. In somefurther embodiments, the cleaning compositions comprise at least 0.0001weight percent of at least one subtilisin variant provided herein andoptionally, a suitable adjunct ingredient.

The present invention also provides methods for cleaning, comprising thesteps of: contacting a surface and/or an article comprising a fabricwith the cleaning compositions provided herein; and optionally washingand/or rinsing the surface or article.

The present invention further provides animal feeds comprising at leastone subtilisin variant provided herein. The present invention alsoprovides food processing compositions comprising at least one subtilisinvariant provided herein.

DESCRIPTION OF THE INVENTION

The present invention provides variant subtilisins and compositionscomprising at least one variant subtilisin set forth herein, as well asmethods for using these variants and compositions. In particular, thepresent invention provides variant proteases suitable for laundrycleaning applications.

In some embodiments, the present invention provides means to producevariant subtilisins with various commercial applications wheredegradation or synthesis of polypeptides are desired, including cleaningcompositions, as well as feed components, textile processing, leatherfinishing, grain processing, meat processing, food processing,preparation of protein hydrolysates, digestive aids, microbicidalcompositions, bacteriostatic compositions, fungistatic compositions, andpersonal care products (e.g., oral care, hair care, and/or skin care).

The present invention further provides enzyme compositions havecomparable or improved wash performance, as compared to presently usedsubtilisin proteases. In some embodiments, the present inventionprovides cleaning compositions comprising at least one subtilisinvariant provided herein. In some embodiments, the cleaning compositionis a laundry detergent. In some embodiments, the laundry detergent is acold water detergent, a low pH detergent, or a compact detergent. Inadditional embodiments, the present invention provides methods forcleaning, comprising the step of contacting a surface and/or an articlecomprising a fabric with a cleaning composition comprising at least onesubtilisin variant.

Cleaning Compositions

The cleaning compositions of the present invention are advantageouslyemployed for example, in laundry applications. Indeed, due to the uniqueadvantages of increased effectiveness in lower temperature solutions,the enzymes of the present invention are ideally suited for laundryapplications. Furthermore, the enzymes of the present invention may beemployed in both granular and liquid compositions.

The variant proteases of the present invention also find use laundrycleaning additive products. In some embodiments, low temperaturesolution cleaning applications find use. The additive product may be, inits simplest form, one or more proteases. In some embodiments, theadditive is packaged in dosage form for addition to a cleaning process.Any suitable single dosage form also finds use with the presentinvention, including but not limited to pills, tablets, gelcaps, orother single dosage units such as pre-measured powders or liquids. Insome embodiments, filler(s) or carrier material(s) are included toincrease the volume of such composition. Suitable filler or carriermaterials include, but are not limited to, various salts of sulfate,carbonate and silicate as well as talc, clay and the like. Suitablefiller or carrier materials for liquid compositions include, but are notlimited to water or low molecular weight primary and secondary alcoholsincluding polyols and diols. Examples of such alcohols include, but arenot limited to, methanol, ethanol, propanol and isopropanol. In someembodiments, the compositions contain from about 5% to about 90% of suchmaterials. Acidic fillers find use to reduce pH. Alternatively, thecleaning additive includes adjunct ingredients as more fully describedbelow.

The present cleaning compositions and cleaning additives require aneffective amount of at least one of the protease variants providedherein, alone or in combination with other proteases and/or additionalenzymes. The required level of enzyme is achieved by the addition of oneor more protease variants of the present invention. Typically thepresent cleaning compositions comprise at least about 0.0001 weightpercent, from about 0.0001 to about 1, from about 0.001 to about 0.5, oreven from about 0.01 to about 0.1 weight percent of at least one of thevariant proteases of the present invention.

The cleaning compositions herein are typically formulated such that,during use in aqueous cleaning operations, the wash water will have a pHof from about 5.0 to about 11.5 or even from about 7.5 to about 10.5.Liquid product formulations are typically formulated to have a neat pHfrom about 3.0 to about 9.0 or even from about 3 to about 5. Granularlaundry products are typically formulated to have a pH from about 9 toabout 11. Techniques for controlling pH at recommended usage levelsinclude the use of buffers, alkalis, acids, etc., and are well known tothose skilled in the art.

Suitable low pH cleaning compositions typically have a neat pH of fromabout 3 to about 5, and are typically free of surfactants that hydrolyzein such a pH environment. Such surfactants include sodium alkyl sulfatesurfactants that comprise at least one ethylene oxide moiety or evenfrom about 1 to about 16 moles of ethylene oxide. Such cleaningcompositions typically comprise a sufficient amount of a pH modifier,such as sodium hydroxide, monoethanolamine or hydrochloric acid, toprovide such cleaning composition with a neat pH of from about 3 toabout 5. Such compositions typically comprise at least one acid stableenzyme. In some embodiments, the compositions are liquids, while inother embodiments, they are solids. The pH of such liquid compositionsis typically measured as a neat pH. The pH of such solid compositions ismeasured as a 10% solids solution of said composition wherein thesolvent is distilled water. In these embodiments, all pH measurementsare taken at 20° C.

In some embodiments, when the variant protease(s) is/are employed in agranular composition or liquid, it is desirable for the variant proteaseto be in the form of an encapsulated particle to protect the variantprotease from other components of the granular composition duringstorage. In addition, encapsulation is also a means of controlling theavailability of the variant protease during the cleaning process. Insome embodiments, encapsulation enhances the performance of the variantprotease(s) and/or additional enzymes. In this regard, the variantproteases of the present invention are encapsulated with any suitableencapsulating material known in the art. In some embodiments, theencapsulating material typically encapsulates at least part of thecatalyst for the variant protease(s) of the present invention.Typically, the encapsulating material is water-soluble and/orwater-dispersible. In some embodiments, the encapsulating material has aglass transition temperature (Tg) of 0° C. or higher. Glass transitiontemperature is described in more detail in WO 97/11151. Theencapsulating material is selected from consisting of carbohydrates,natural or synthetic gums, chitin, chitosan, cellulose and cellulosederivatives, silicates, phosphates, borates, polyvinyl alcohol,polyethylene glycol, paraffin waxes, and combinations thereof. When theencapsulating material is a carbohydrate, it is typically selected frommonosaccharides, oligosaccharides, polysaccharides, and combinationsthereof. Typically, the encapsulating material is a starch. Suitablestarches are described in EP 0 922 499; U.S. Pat. No. 4,977,252; U.S.Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826. In some embodiments,the encapsulating material is a microsphere made from plastic such asthermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile,polymethacrylonitrile and mixtures thereof; commercially availablemicrospheres that find use include those supplied by EXPANCEL®(Stockviksverken, Sweden), and PM 6545, PM 6550, PM 7220, PM 7228,EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., ValleyForge, Pa.)>

As described herein, the variant proteases of the present invention findparticular use in laundry detergents. These applications place enzymesunder various environmental stresses. The variant proteases of thepresent invention provide advantages over many currently used enzymes,due to their stability under various conditions.

Indeed, there are a variety of wash conditions including varyingdetergent formulations, wash water volumes, wash water temperatures, andlengths of wash time, to which proteases involved in washing areexposed. In addition, detergent formulations used in differentgeographical areas have different concentrations of their relevantcomponents present in the wash water. For example, a European detergenttypically has about 4500-5000 ppm of detergent components in the washwater, while a Japanese detergent typically has approximately 667 ppm ofdetergent components in the wash water. In North America, particularlythe United States, detergents typically have about 975 ppm of detergentcomponents present in the wash water.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water. Brazil typicallyhas approximately 1500 ppm of detergent components present in the washwater.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S. and about1500 ppm in Brazil, to greater than about 2000 ppm (“high detergentconcentration geographies”), for example about 4500 ppm to about 5000ppm in Europe and about 6000 ppm in high suds phosphate buildergeographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solutionabout 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature of the washwater in North America and Japan is typically between 10 and 30° C.(e.g., about 20° C.), whereas the temperature of wash water in Europe istypically between 30 and 60° C. (e.g., about 40° C.).

As a further example, different geographies typically have differentwater hardness. Water hardness is usually described in terms of thegrains per gallon mixed Ca²⁺/Mg²⁺. Hardness is a measure of the amountof calcium (Ca²⁺) and magnesium (Mg²⁺) in the water. Most water in theUnited States is hard, but the degree of hardness varies. Moderatelyhard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts permillion (parts per million converted to grains per U.S. gallon is ppm #divided by 17.1 equals grains per gallon) of hardness minerals.

Water Grains per gallon Parts per million Soft less than 1.0 less than17 Slightly hard 1.0 to 3.5 17 to 60 Moderately hard 3.5 to 7.0 60 to120 Hard 7.0 to 10.5 120 to 180 Very hard greater than 10.5 greater than180

European water hardness is typically greater than 10.5 (for example10.5-20.0) grains per gallon mixed Ca²⁺/Mg²⁺ (e.g., about 15 grains pergallon mixed Ca²⁺/Mg²⁺). North American water hardness is typicallygreater than Japanese water hardness, but less than European waterhardness. For example, North American water hardness can be between 3 to10 grains, 3-8 grains or about 6 grains. Japanese water hardness istypically lower than North American water hardness, usually less than 4,for example 3 grains per gallon mixed Ca²⁺/Mg²⁺.

Accordingly, in some embodiments, the present invention provides variantproteases that show surprising wash performance in at least one set ofwash conditions (e.g., water temperature, water hardness, and/ordetergent concentration). In some embodiments, the variant proteases ofthe present invention are comparable in wash performance to othersubtilisin proteases. In some embodiments, the variant proteases of thepresent invention exhibit enhanced wash performance as compared tosubtilisin proteases currently commercially available. Thus, in somepreferred embodiments of the present invention, the variant proteasesprovided herein exhibit enhanced oxidative stability, enhanced thermalstability, and/or enhanced chelator stability. In addition, the variantproteases of the present invention find use in cleaning compositionsthat do not include detergents, again either alone or in combinationwith builders and stabilizers.

In some embodiments of the present invention, the cleaning compositionscomprise at least one variant protease of the present invention at alevel from about 0.00001% to about 10% by weight of the composition andthe balance (e.g., about 99.999% to about 90.0%) comprising cleaningadjunct materials by weight of composition. In other aspects of thepresent invention, the cleaning compositions of the present inventioncomprises at least one variant protease at a level of about 0.0001% toabout 10%, about 0.001% to about 5%, about 0.001% to about 2%, about0.005% to about 0.5% by weight of the composition and the balance of thecleaning composition (e.g., about 99.9999% to about 90.0%, about 99.999%to about 98%, about 99.995% to about 99.5% by weight) comprisingcleaning adjunct materials.

In some embodiments, preferred cleaning compositions comprise one ormore additional enzymes or enzyme derivatives which provide cleaningperformance and/or fabric care benefits, in addition to one or more ofthe variant proteases provided herein. Such enzymes include, but are notlimited to other proteases, lipases, cutinases, amylases, cellulases,peroxidases, oxidases (e.g. laccases), and/or mannanases.

Any other suitable protease finds use in the compositions of the presentinvention. Suitable proteases include those of animal, vegetable ormicrobial origin. In some particularly preferred embodiments, microbialproteases are used. In some embodiments, chemically or geneticallymodified mutants are included. In some embodiments, the protease is aserine protease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases includesubtilisins, especially those derived from Bacillus (e.g., subtilisin,lentus, amyloliquefaciens, subtilisin Carlsberg, subtilisin 309,subtilisin 147 and subtilisin 168). Additional examples include thosemutant proteases described in U.S. Pat. Nos. RE 34,606, 5,955,340,5,700,676, 6,312,936, and 6,482,628, all of which are incorporatedherein by reference. Additional protease examples include, but are notlimited to trypsin (e.g., of porcine or bovine origin), and the Fusariumprotease described in WO 89/06270. Preferred commercially availableprotease enzymes include MAXATASE®, MAXACAL™, MAXAPEM™, OPTICLEAN®,OPTIMASE®, PROPERASE®, PURAFECT® and PURAFECT® OXP (Genencor);ALCALASE®, SAVINASE®, PRIMASE®, DURAZYM™, RELASE® and ESPERASE®(Novozymes); and BLAP™ (Henkel Kommanditgesellschaft auf Aktien,Duesseldorf, Germany. Various proteases are described in WO95/23221, WO92/21760, and U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625,U.S. Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and6,482,628, and various other patents.

In addition, any suitable lipase finds use in the present invention.Suitable lipases include, but are not limited to those of bacterial orfungal origin. Chemically or genetically modified mutants areencompassed by the present invention. Examples of useful lipases includeHumicola lanuginosa lipase (See e.g., EP 258 068, and EP 305 216),Rhizomucor miehei lipase (See e.g., EP 238 023), Candida lipase, such asC. antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g.,EP 214 761), a Pseudomonas lipase such as P. alcaligenes and P.pseudoalcaligenes lipase (See e.g., EP 218 272), P. cepacia lipase (Seee.g., EP 331 376), P. stutzeri lipase (See e.g., GB 1,372,034), P.fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartoiset al., Biochem. Biophys. Acta 1131:253-260 [1993]); B.stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumiluslipase [See e.g., WO 91/16422]).

Furthermore, a number of cloned lipases find use in some embodiments ofthe present invention, including but not limited to Penicilliumcamembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]),Geotricum candidum lipase (See, Schimada et al., J. Biochem.,106:383-388 [1989]), and various Rhizopus lipases such as R. delemarlipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase(Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R.oryzae lipase.

Other types of lipolytic enzymes such as cutinases also find use in someembodiments of the present invention, including but not limited to thecutinase derived from Pseudomonas mendocina (See, WO 88/09367), and thecutinase derived from Fusarium solani pisi (See, WO 90/09446).

Additional suitable lipases include commercially available lipases suchas M1 LIPASE™, LUMA FAST™, and LIPOMAX™ (Genencor); LIPOLASE® andLIPOLASE® ULTRA (Novozymes); and LIPASE P™ “Amano” (Amano PharmaceuticalCo. Ltd., Japan).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise lipases at a level from about0.00001% to about 10% of additional lipase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In other aspects of the present invention, the cleaning compositions ofthe present invention also comprise, lipases at a level of about 0.0001%to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about0.005% to about 0.5% lipase by weight of the composition.

Any amylase (alpha and/or beta) suitable for use in alkaline solutionsalso find use in some embodiments of the present invention. Suitableamylases include, but are not limited to those of bacterial or fungalorigin. Chemically or genetically modified mutants are included in someembodiments. Amylases that find use in the present invention, include,but are not limited to α-amylases obtained from B. licheniformis (Seee.g., GB 1,296,839). Commercially available amylases that find use inthe present invention include, but are not limited to DURAMYL®,TERMAMYL®, FUNGAMYL® and BAN™ (Novozymes) and RAPIDASE® and MAXAMYL® P(Genencor).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise amylases at a level from about0.00001% to about 10% of additional amylase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In other aspects of the present invention, the cleaning compositions ofthe present invention also comprise, amylases at a level of about0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about2%, about 0.005% to about 0.5% amylase by weight of the composition.

In some further embodiments, any suitable cellulase finds used in thecleaning compositions of the present invention. Suitable cellulasesinclude, but are not limited to those of bacterial or fungal origin.Chemically or genetically modified mutants are included in someembodiments. Suitable cellulases include, but are not limited toHumicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307).Especially suitable cellulases are the cellulases having color carebenefits (See e.g., EP 0 495 257). Commercially available cellulasesthat find use in the present include, but are not limited to CELLUZYME®(Novozymes), and KAC-500(B)™ (Kao Corporation). In some embodiments,cellulases are incorporated as portions or fragments of mature wild-typeor variant cellulases, wherein a portion of the N-terminus is deleted(See e.g., U.S. Pat. No. 5,874,276). In some embodiments, the cleaningcompositions of the present invention further comprise cellulases at alevel from about 0.00001% to about 10% of additional cellulase by weightof the composition and the balance of cleaning adjunct materials byweight of composition. In other aspects of the present invention, thecleaning compositions of the present invention also comprise cellulasesat a level of about 0.0001% to about 10%, about 0.001% to about 5%,about 0.001% to about 2%, about 0.005% to about 0.5% cellulase by weightof the composition.

Any mannanase suitable for use in detergent compositions also finds usein the present invention. Suitable mannanases include, but are notlimited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Variousmannanases are known which find use in the present invention (See e.g.,U.S. Pat. No. 6,566,114, U.S. Pat. No. 6,602,842, and U.S. Pat. No.6,440,991, all of which are incorporated herein by reference). In someembodiments, the cleaning compositions of the present invention furthercomprise mannanases at a level from about 0.00001% to about 10% ofadditional mannanase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In other aspects ofthe present invention, the cleaning compositions of the presentinvention also comprise, mannanases at a level of about 0.0001% to about10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% toabout 0.5% mannanase by weight of the composition.

In some embodiments, peroxidases are used in combination with hydrogenperoxide or a source thereof (e.g., a percarbonate, perborate orpersulfate) in the compositions of the present invention. In somealternative embodiments, oxidases are used in combination with oxygen.Both types of enzymes are used for “solution bleaching” (i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen the fabrics are washed together in a wash liquor), preferablytogether with an enhancing agent (See e.g., WO 94/12621 and WO95/01426). Suitable peroxidases/oxidases include, but are not limited tothose of plant, bacterial or fungal origin. Chemically or geneticallymodified mutants are included in some embodiments. In some embodiments,the cleaning compositions of the present invention further compriseperoxidase and/or oxidase enzymes at a level from about 0.00001% toabout 10% of additional peroxidase and/or oxidase by weight of thecomposition and the balance of cleaning adjunct materials by weight ofcomposition. In other aspects of the present invention, the cleaningcompositions of the present invention also comprise peroxidase and/oroxidase enzymes at a level of about 0.0001% to about 10%, about 0.001%to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5%peroxidase and/or oxidase enzymes by weight of the composition.

In some embodiments, additional enzymes find use, including but notlimited to perhydrolases (See e.g., WO 05/056782). In addition, in someparticularly preferred embodiments, mixtures of the above mentionedenzymes are encompassed herein, in particular one or more additionalprotease, amylase, lipase, mannanase, and/or at least one cellulase.Indeed, it is contemplated that various mixtures of these enzymes willfind use in the present invention. It is also contemplated that thevarying levels of the variant protease(s) and one or more additionalenzymes may both independently range to about 10%, the balance of thecleaning composition being cleaning adjunct materials. The specificselection of cleaning adjunct materials are readily made by consideringthe surface, item, or fabric to be cleaned, and the desired form of thecomposition for the cleaning conditions during use (e.g., through thewash detergent use).

Examples of suitable cleaning adjunct materials include, but are notlimited to, surfactants, builders, bleaches, bleach activators, bleachcatalysts, other enzymes, enzyme stabilizing systems, chelants, opticalbrighteners, soil release polymers, dye transfer agents, dispersants,suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes,photoactivators, fluorescers, fabric conditioners, hydrolyzablesurfactants, preservatives, anti-oxidants, anti-shrinkage agents,anti-wrinkle agents, germicides, fungicides, color speckles, silvercare,anti-tarnish and/or anti-corrosion agents, alkalinity sources,solubilizing agents, carriers, processing aids, pigments, and pH controlagents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464,5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of whichare incorporated herein by reference). Embodiments of specific cleaningcomposition materials are exemplified in detail below. In embodiments inwhich the cleaning adjunct materials are not compatible with the variantproteases of the present invention in the cleaning compositions, thensuitable methods of keeping the cleaning adjunct materials and theprotease(s) separated (i.e., not in contact with each other) untilcombination of the two components is appropriate are used. Suchseparation methods include any suitable method known in the art (e.g.,gelcaps, encapulation, tablets, physical separation, etc.).

By way of example, several cleaning compositions wherein the variantproteases of the present invention find use are described in greaterdetail below. In embodiments in which the cleaning compositions of thepresent invention are formulated as compositions suitable for use inlaundry machine washing method(s), the compositions of the presentinvention preferably contain at least one surfactant and at least onebuilder compound, as well as one or more cleaning adjunct materialspreferably selected from organic polymeric compounds, bleaching agents,additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. In some embodiments, laundry compositions also containsoftening agents (i.e., as additional cleaning adjunct materials). Thecompositions of the present invention also find use detergent additiveproducts in solid or liquid form. Such additive products are intended tosupplement and/or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process. Insome embodiments, the density of the laundry detergent compositionsherein ranges from about 400 to about 1200 g/liter, while in otherembodiments, it ranges from about 500 to about 950 g/liter ofcomposition measured at 20° C.

In some embodiments, various cleaning compositions such as thoseprovided in U.S. Pat. No. 6,605,458 find use with the variant proteasesof the present invention. Thus, in some embodiments, the compositionscomprising at least one variant protease of the present invention is acompact granular fabric cleaning composition, while in otherembodiments, the composition is a granular fabric cleaning compositionuseful in the laundering of colored fabrics, in further embodiments, thecomposition is a granular fabric cleaning composition which providessoftening through the wash capacity, in additional embodiments, thecomposition is a heavy duty liquid fabric cleaning composition. In someembodiments, the compositions comprising at least one variant proteaseof the present invention are fabric cleaning compositions such as thosedescribed in U.S. Pat. Nos. 6,610,642 and 6,376,450. In addition, thevariant proteases of the present invention find use in granular laundrydetergent compositions of particular utility under European or Japanesewashing conditions (See e.g., U.S. Pat. No. 6,610,642).

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in U.S. Pat. Nos.5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference. When a low pH cleaning composition is desired, the pH of suchcomposition is adjusted via the addition of a material such asmonoethanolamine or an acidic material such as HCl.

While not essential for the purposes of the present invention, thenon-limiting list of adjuncts illustrated hereinafter are suitable foruse in the instant cleaning compositions. In some embodiments, theseadjuncts are incorporated for example, to assist or enhance cleaningperformance, for treatment of the substrate to be cleaned, or to modifythe aesthetics of the cleaning composition as is the case with perfumes,colorants, dyes or the like. It is understood that such adjuncts are inaddition to the variant proteases of the present invention. The precisenature of these additional components, and levels of incorporationthereof, will depend on the physical form of the composition and thenature of the cleaning operation for which it is to be used. Suitableadjunct materials include, but are not limited to, surfactants,builders, chelating agents, dye transfer inhibiting agents, depositionaids, dispersants, additional enzymes, and enzyme stabilizers, catalyticmaterials, bleach activators, bleach boosters, hydrogen peroxide,sources of hydrogen peroxide, preformed peracids, polymeric dispersingagents, clay soil removal/anti-redeposition agents, brighteners, sudssuppressors, dyes, perfumes, structure elasticizing agents, fabricsofteners, carriers, hydrotropes, processing aids and/or pigments. Inaddition to the disclosure below, suitable examples of such otheradjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282,6,306,812, and 6,326,348, that are incorporated by reference. Theaforementioned adjunct ingredients may constitute the balance of thecleaning compositions of the present invention.

In some embodiments, the cleaning compositions according to the presentinvention comprise a surfactant or surfactant system wherein thesurfactant is selected from nonionic surfactants, anionic surfactants,cationic surfactants, ampholytic surfactants, zwitterionic surfactants,semi-polar nonionic surfactants and mixtures thereof.

In some additional embodiments, the cleaning compositions of the presentinvention comprise one or more detergent builders or builder systems.Builders include, but are not limited to, the alkali metal, ammonium andalkanolammonium salts of polyphosphates, alkali metal silicates,alkaline earth and alkali metal carbonates, aluminosilicate builderspolycarboxylate compounds. ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, thevarious alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof.

In some further embodiments, the cleaning compositions herein contain achelating agent. Suitable chelating agents include copper, iron and/ormanganese chelating agents and mixtures thereof.

In some still further embodiments, the cleaning compositions providedherein contain a deposition aid. Suitable deposition aids include,polyethylene glycol, polypropylene glycol, polycarboxylate, soil releasepolymers such as polytelephthalic acid, clays such as Kaolinite,montmorillonite, atapulgite, illite, bentonite, halloysite, and mixturesthereof.

In some additional embodiments, the cleaning compositions of the presentinvention also include one or more dye transfer inhibiting agents.Suitable polymeric dye transfer inhibiting agents include, but are notlimited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers,copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.

In some still additional embodiments, the cleaning compositions of thepresent invention also contain dispersants. Suitable water-solubleorganic materials include the homo- or co-polymeric acids or theirsalts, in which the polycarboxylic acid comprises at least two carboxylradicals separated from each other by not more than two carbon atoms.

In some particularly preferred embodiments, the cleaning compositionscomprise one or more detergent enzymes which provide cleaningperformance and/or fabric care benefits. Examples of suitable enzymesinclude, but are not limited to, hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, keratinases, reductases, oxidases, phenol oxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. A typical combination is acocktail of conventional applicable enzymes including at least oneprotease, at least one lipase, at least one cutinase, and/or at leastone cellulase in conjunction with at least one amylase.

In some further embodiments, the enzymes used in the cleaningcompositions are stabilized any suitable technique. In some embodiments,the enzymes employed herein are stabilized by the presence ofwater-soluble sources of calcium and/or magnesium ions in the finishedcompositions that provide such ions to the enzymes.

In some still further embodiments, the cleaning compositions of thepresent invention include catalytic metal complexes. One type ofmetal-containing bleach catalyst is a catalyst system comprising atransition metal cation of defined bleach catalytic activity, such ascopper, iron, titanium, ruthenium, tungsten, molybdenum, or manganesecations, an auxiliary metal cation having little or no bleach catalyticactivity, such as zinc or aluminum cations, and a sequestrate havingdefined stability constants for the catalytic and auxiliary metalcations, particularly ethylenediaminetetraacetic acid,ethylenediaminetetra (methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.

In some embodiments, the compositions herein are catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, for example, the manganese-based catalystsdisclosed in U.S. Pat. No. 5,576,282. In addition, cobalt bleachcatalysts useful herein are known, and are described, for example, inU.S. Pat. Nos. 5,597,936, and 5,595,967. Such cobalt catalysts arereadily prepared by known procedures, such as taught for example in U.S.Pat. Nos. 5,597,936, and 5,595,967. In some embodiments, thecompositions provided herein also suitably include a transition metalcomplex of a macropolycyclic rigid ligand (i.e., “MRL”). As a practicalmatter, and not by way of limitation, the compositions and cleaningprocesses herein are adjustable, to provide on the order of at least onepart per hundred million of the active MRL species in the aqueouswashing medium, and will preferably provide from about 0.005 ppm toabout 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, andmost preferably from about 0.1 ppm to about 5 ppm, of the MRL in thewash liquor. Preferred transition-metals in the instant transition-metalbleach catalyst include manganese, iron and chromium. Preferred MRLsherein are a special type of ultra-rigid ligand that is cross-bridgedsuch as 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught for example in WO 00/332601, and U.S. Pat. No. 6,225,464.

As indicated above, the cleaning compositions of the present inventionare formulated into any suitable form and prepared by any process chosenby the formulator, non-limiting examples of which are described in U.S.Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference.

The cleaning compositions disclosed herein of find use in cleaning asitus (e.g., a surface, dishware, or fabric). Typically, at least aportion of the situs is contacted with an embodiment of the presentcleaning composition, in neat form or diluted in a wash liquor, and thenthe situs is optionally washed and/or rinsed. For purposes of thepresent invention, “washing” includes but is not limited to, scrubbing,and mechanical agitation. In some embodiments, the cleaning compositionsare typically employed at concentrations of from about 500 ppm to about15,000 ppm in solution. When the wash solvent is water, the watertemperature typically ranges from about 5° C. to about 90° C. and, whenthe situs comprises a fabric, the water to fabric mass ratio istypically from about 1:1 to about 30:1.

DEFINITIONS

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,protein engineering, microbiology, and recombinant DNA, which are withinthe skill of the art. Such techniques are known to those of skill in theart and are described in numerous texts and reference works (See e.g.,Sambrook et al., “Molecular Cloning: A Laboratory Manual”, SecondEdition (Cold Spring Harbor), [1989]); and Ausubel et al., “CurrentProtocols in Molecular Biology” [1987]). All patents, patentapplications, articles and publications mentioned herein, both supra andinfra, are hereby expressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. There are variousdictionaries available and known to those in the art that providedefinitions of these terms. Although any methods and materials similaror equivalent to those described herein find use in the practice of thepresent invention, the preferred methods and materials are describedherein. Accordingly, the terms defined immediately below are more fullydescribed by reference to the Specification as a whole. Also, as usedherein, the singular “a”, “an” and “the” includes the plural referenceunless the context clearly indicates otherwise. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. It is to be understood that this invention isnot limited to the particular methodology, protocols, and reagentsdescribed, as these may vary, depending upon the context they are usedby those of skill in the art.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of protein purification, molecularbiology, microbiology, recombinant DNA techniques and proteinsequencing, all of which are within the skill of those in the art.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

As used herein, the terms “protease,” and “proteolytic activity” referto a protein or peptide exhibiting the ability to hydrolyze peptides orsubstrates having peptide linkages. Many well known procedures exist formeasuring proteolytic activity (Kalisz, “Microbial Proteinases,” In:Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology,[1988]). For example, proteolytic activity may be ascertained bycomparative assays which analyze the respective protease's ability tohydrolyze a commercial substrate. Exemplary substrates useful in theanalysis of protease or proteolytic activity, include, but are notlimited to di-methyl casein (Sigma C-9801), bovine collagen (SigmaC-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICNBiomedical 902111). Colorimetric assays utilizing these substrates arewell known in the art (See e.g., WO 99/34011; and U.S. Pat. No.6,376,450, both of which are incorporated herein by reference). The pNAassay (See e.g., Del Mar et al., Anal. Biochem., 99:316-320 [1979]) alsofinds use in determining the active enzyme concentration for fractionscollected during gradient elution. This assay measures the rate at whichp-nitroaniline is released as the enzyme hydrolyzes the solublesynthetic substrate,succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide(sAAPF-pNA). The rate of production of yellow color from the hydrolysisreaction is measured at 410 nm on a spectrophotometer and isproportional to the active enzyme concentration. In addition, absorbancemeasurements at 280 nm can be used to determine the total proteinconcentration. The active enzyme/total-protein ratio gives the enzymepurity.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include, but arenot limited to, a single-, double- or triple-stranded DNA, genomic DNA,cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically, biochemically modified, non-naturalor derivatized nucleotide bases. The following are non-limiting examplesof polynucleotides: genes, gene fragments, chromosomal fragments, ESTs,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. In some embodiments, polynucleotides comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. In alternative embodiments, thesequence of nucleotides is interrupted by non-nucleotide components.

As used herein, the terms “DNA construct” and “transforming DNA” areused interchangeably to refer to DNA used to introduce sequences into ahost cell or organism. The DNA may be generated in vitro by PCR or anyother suitable technique(s) known to those in the art. In particularlypreferred embodiments, the DNA construct comprises a sequence ofinterest (e.g., as an incoming sequence). In some embodiments, thesequence is operably linked to additional elements such as controlelements (e.g., promoters, etc.). The DNA construct may further comprisea selectable marker. It may further comprise an incoming sequenceflanked by homology boxes. In a further embodiment, the transforming DNAcomprises other non-homologous sequences, added to the ends (e.g.,stuffer sequences or flanks). In some embodiments, the ends of theincoming sequence are closed such that the transforming DNA forms aclosed circle. The transforming sequences may be wild-type, mutant ormodified. In some embodiments, the DNA construct comprises sequenceshomologous to the host cell chromosome. In other embodiments, the DNAconstruct comprises non-homologous sequences. Once the DNA construct isassembled in vitro it may be used to: 1) insert heterologous sequencesinto a desired target sequence of a host cell, and/or 2) mutagenize aregion of the host cell chromosome (i.e., replace an endogenous sequencewith a heterologous sequence), 3) delete target genes, and/or 4)introduce a replicating plasmid into the host.

As used herein, the terms “expression cassette” and “expression vector”refer to nucleic acid constructs generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a target cell. Therecombinant expression cassette can be incorporated into a plasmid,chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acidfragment. Typically, the recombinant expression cassette portion of anexpression vector includes, among other sequences, a nucleic acidsequence to be transcribed and a promoter. In preferred embodiments,expression vectors have the ability to incorporate and expressheterologous DNA fragments in a host cell. Many prokaryotic andeukaryotic expression vectors are commercially available. Selection ofappropriate expression vectors is within the knowledge of those of skillin the art. The term “expression cassette” is used interchangeablyherein with “DNA construct,” and their grammatical equivalents.Selection of appropriate expression vectors is within the knowledge ofthose of skill in the art.

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmids,cassettes and the like. In some embodiments, the polynucleotideconstruct comprises a DNA sequence encoding the protease (e.g.,precursor or mature protease) that is operably linked to a suitableprosequence (e.g., secretory, etc.) capable of effecting the expressionof the DNA in a suitable host.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes orprokaryotes, or integrates into the host chromosome.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, conjugation, and transduction (See e.g.,Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus, PlenumPublishing Corp., pages 57-72, [1989]).

As used herein, the terms “transformed” and “stably transformed” refersto a cell that has a non-native (heterologous) polynucleotide sequenceintegrated into its genome or as an episomal plasmid that is maintainedfor at least two generations.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader (i.e., a signal peptide), is operably linkedto DNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice.

As used herein the term “gene” refers to a polynucleotide (e.g., a DNAsegment), that encodes a polypeptide and includes regions preceding andfollowing the coding regions as well as intervening sequences (introns)between individual coding segments (exons).

As used herein, “homologous genes” refers to a pair of genes fromdifferent, but usually related species, which correspond to each otherand which are identical or very similar to each other. The termencompasses genes that are separated by speciation (i.e., thedevelopment of new species) (e.g., orthologous genes), as well as genesthat have been separated by genetic duplication (e.g., paralogousgenes).

As used herein, proteins are defined as having a common “fold” if theyhave the same major secondary structures in the same arrangement andwith the same topological connections. Different proteins with the samefold often have peripheral elements of secondary structure and turnregions that differ in size and conformation. In some cases, thesediffering peripheral regions may comprise half the structure. Proteinsplaced together in the same fold category do not necessarily have acommon evolutionary origin (e.g., structural similarities arising fromthe physics and chemistry of proteins favoring certain packingarrangements and chain topologies).

As used herein, “homology” refers to sequence similarity or identity,with identity being preferred. This homology is determined usingstandard techniques known in the art (See e.g., Smith and Waterman, Adv.Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443[1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988];programs such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res., 12:387-395 [1984]).

As used herein, an “analogous sequence” is one wherein the function ofthe gene is essentially the same as the gene based on the wild-typesubtilisin protease. Additionally, analogous genes include at leastabout 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%,about 99%, or about 100% sequence identity with the sequence of thewild-type subtilisin protease. Alternately, analogous sequences have analignment of between about 70 to about 100% of the genes found in the B.amyloliquefaciens subtilisin protease region. In additional embodimentsmore than one of the above properties applies to the sequence. Analogoussequences are determined by known methods of sequence alignment. Acommonly used alignment method is BLAST, although as indicated above andbelow, there are other methods that also find use in aligning sequences.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pair-wise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng and Doolittle (Feng andDoolittle, J. Mol. Evol., 35:351-360 [1987]). The method is similar tothat described by Higgins and Sharp (Higgins and Sharp, CABIOS 5:151-153[1989]). Useful PILEUP parameters including a default gap weight of3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedby Altschul et al., (Altschul et al., J. Mol. Biol., 215:403-410,[1990]; and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787[1993]). A particularly useful BLAST program is the WU-BLAST-2 program(See, Altschul et al., Meth. Enzymol., 266:460-480 [1996]). WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched. However, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored).

Thus, “percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues of the starting sequence (i.e.,the sequence of interest). A preferred method utilizes the BLASTN moduleof WU-BLAST-2 set to the default parameters, with overlap span andoverlap fraction set to 1 and 0.125, respectively.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention. “Recombination,” “recombining,” and generating a“recombined” nucleic acid are generally the assembly of two or morenucleic acid fragments wherein the assembly gives rise to a chimericgene.

In some preferred embodiments, mutant DNA sequences are generated withsite saturation mutagenesis in at least one codon. In another preferredembodiment, site saturation mutagenesis is performed for two or morecodons. In a further embodiment, mutant DNA sequences have more thanabout 50%, more than about 55%, more than about 60%, more than about65%, more than about 70%, more than about 75%, more than about 80%, morethan about 85%, more than about 90%, more than about 95%, or more thanabout 98% homology with the wild-type sequence. In alternativeembodiments, mutant DNA is generated in vivo using any known mutagenicprocedure such as, for example, radiation, nitrosoguanidine and thelike. The desired DNA sequence is then isolated and used in the methodsprovided herein.

As used herein, the terms “amplification” and “gene amplification” referto a process by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. In some embodiments,selection of cells by growth in the presence of a drug (e.g., aninhibitor of an inhibitable enzyme) results in the amplification ofeither the endogenous gene encoding the gene product required for growthin the presence of the drug or by amplification of exogenous (i.e.,input) sequences encoding this gene product, or both.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods of U.S. Pat. Nos. 4,683,195 4,683,202, and 4,965,188, herebyincorporated by reference, which include methods for increasing theconcentration of a segment of a target sequence in a mixture of genomicDNA without cloning or purification. This process for amplifying thetarget sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the “polymerase chainreaction” (hereinafter “PCR”). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the term “RT-PCR” refers to the replication andamplification of RNA sequences. In this method, reverse transcription iscoupled to PCR, most often using a one enzyme procedure in which athermostable polymerase is employed, as described in U.S. Pat. No.5,322,770, herein incorporated by reference. In RT-PCR, the RNA templateis converted to cDNA due to the reverse transcriptase activity of thepolymerase, and then amplified using the polymerizing activity of thepolymerase (i.e., as in other PCR methods).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A “restriction site” refers to a nucleotide sequence recognized andcleaved by a given restriction endonuclease and is frequently the sitefor insertion of DNA fragments. In certain embodiments of the inventionrestriction sites are engineered into the selective marker and into 5′and 3′ ends of the DNA construct.

“Homologous recombination” means the exchange of DNA fragments betweentwo DNA molecules or paired chromosomes at the site of identical ornearly identical nucleotide sequences. In a preferred embodiment,chromosomal integration is homologous recombination.

As used herein “amino acid” refers to peptide or protein sequences orportions thereof. The terms “protein,” “peptide,” and “polypeptide” areused interchangeably.

As used herein, “protein of interest” and “polypeptide of interest”refer to a protein/polypeptide that is desired and/or being assessed. Insome embodiments, the protein of interest is expressed intracellularly,while in other embodiments, it is a secreted polypeptide. Inparticularly preferred embodiments, these enzymes include the serineproteases of the present invention. In some embodiments, the protein ofinterest is a secreted polypeptide which is fused to a signal peptide(i.e., an amino-terminal extension on a protein to be secreted). Nearlyall secreted proteins use an amino-terminal protein extension whichplays a crucial role in the targeting to and translocation of precursorproteins across the membrane. This extension is proteolytically removedby a signal peptidase during or immediately following membrane transfer.

A polynucleotide is said to “encode” an RNA or a polypeptide if, in itsnative state or when manipulated by methods known to those of skill inthe art, it can be transcribed and/or translated to produce the RNA, thepolypeptide or a fragment thereof. The anti-sense strand of such anucleic acid is also said to encode the sequences. As is known in theart, a DNA can be transcribed by an RNA polymerase to produce RNA, butan RNA can be reverse transcribed by reverse transcriptase to produce aDNA. Thus a DNA can encode a RNA and vice versa.

“Host strain” or “host cell” refers to a suitable host for an expressionvector comprising DNA according to the present invention.

An enzyme is “overexpressed” in a host cell if the enzyme is expressedin the cell at a higher level that the level at which it is expressed ina corresponding wild-type cell.

The terms “protein” and “polypeptide” are used interchangeabilityherein. The 3-letter code for amino acids as defined in conformity withthe IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) isused through out this disclosure. It is also understood that apolypeptide may be coded for by more than one nucleotide sequence due tothe degeneracy of the genetic code.

A “prosequence” is an amino acid sequence between the signal sequenceand mature protease that is necessary for the secretion of the protease.Cleavage of the pro sequence results in a mature active protease.

The term “signal sequence” or “signal peptide” refers to any sequence ofnucleotides and/or amino acids which may participate in the secretion ofthe mature or precursor forms of the protein. This definition of signalsequence is a functional one, meant to include all those amino acidsequences encoded by the N-terminal portion of the protein gene, whichparticipate in the effectuation of the secretion of protein. They areoften, but not universally, bound to the N-terminal portion of a proteinor to the N-terminal portion of a precursor protein. The signal sequencemay be endogenous or exogenous. The signal sequence may be that normallyassociated with the protein (e.g., protease), or may be from a geneencoding another secreted protein. One exemplary exogenous signalsequence comprises the first seven amino acid residues of the signalsequence from Bacillus subtilis subtilisin fused to the remainder of thesignal sequence of the subtilisin from Bacillus lentus (ATCC 21536).

The term “hybrid signal sequence” refers to signal sequences in whichpart of sequence is obtained from the expression host fused to thesignal sequence of the gene to be expressed. In some embodiments,synthetic sequences are utilized.

The term “mature” form of a protein or peptide refers to the finalfunctional form of the protein or peptide. For example, the mature formof the BPN′ subtilisin protease of the present invention includes atleast the amino acid sequence identical to residue positions 1-275 ofSEQ ID NO:1, as set forth below:

(SEQ ID NO: 1) AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ 

The term “precursor” form of a protein or peptide refers to a matureform of the protein having a prosequence operably linked to the amino orcarbonyl terminus of the protein. The precursor may also have a “signal”sequence operably linked, to the amino terminus of the prosequence. Theprecursor may also have additional polynucleotides that are involved inpost-translational activity (e.g., polynucleotides cleaved therefrom toleave the mature form of a protein or peptide).

“Naturally occurring enzyme” refers to an enzyme having the unmodifiedamino acid sequence identical to that found in nature. Naturallyoccurring enzymes include native enzymes, those enzymes naturallyexpressed or found in the particular microorganism.

The terms “derived from” and “obtained from” refer to not only aprotease produced or producible by a strain of the organism in question,but also a protease encoded by a DNA sequence isolated from such strainand produced in a host organism containing such DNA sequence.Additionally, the term refers to a protease which is encoded by a DNAsequence of synthetic and/or cDNA origin and which has the identifyingcharacteristics of the protease in question.

A “derivative” within the scope of this definition generally retains thecharacteristic proteolytic activity observed in the wild-type, native orparent form to the extent that the derivative is useful for similarpurposes as the wild-type, native or parent form. Functional derivativesof serine protease encompass naturally occurring, synthetically orrecombinantly produced peptides or peptide fragments which have thegeneral characteristics of the serine protease of the present invention.

The term “functional derivative” refers to a derivative of a nucleicacid which has the functional characteristics of a nucleic acid whichencodes serine protease. Functional derivatives of a nucleic acid whichencode serine protease of the present invention encompass naturallyoccurring, synthetically or recombinantly produced nucleic acids orfragments and encode serine protease characteristic of the presentinvention. Wild type nucleic acid encoding serine proteases according tothe invention include naturally occurring alleles and homologues basedon the degeneracy of the genetic code known in the art.

The term “identical” in the context of two nucleic acids or polypeptidesequences refers to the residues in the two sequences that are the samewhen aligned for maximum correspondence, as measured using one of thefollowing sequence comparison or analysis algorithms.

The term “optimal alignment” refers to the alignment giving the highestpercent identity score.

“Percent sequence identity,” “percent amino acid sequence identity,”“percent gene sequence identity,” and/or “percent nucleicacid/polynucleotide sequence identity,” with respect to two amino acid,polynucleotide and/or gene sequences (as appropriate), refer to thepercentage of residues that are identical in the two sequences when thesequences are optimally aligned. Thus, 80% amino acid sequence identitymeans that 80% of the amino acids in two optimally aligned polypeptidesequences are identical.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides thus refers to a polynucleotide or polypeptide thatcomprising at least about 70% sequence identity, preferably at leastabout 75%, preferably at least about 80%, preferably at least about 85%,preferably at least about 90%, preferably at least about 95%, preferablyat least about 97%, preferably at least about 98%, and preferably atleast about 99% sequence identity as compared to a reference sequenceusing the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) usingstandard parameters. One indication that two polypeptides aresubstantially identical is that the first polypeptide is immunologicallycross-reactive with the second polypeptide. Typically, polypeptides thatdiffer by conservative amino acid substitutions are immunologicallycross-reactive. Thus, a polypeptide is substantially identical to asecond polypeptide, for example, where the two peptides differ only by aconservative substitution. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

The phrase “equivalent” in this context, refers to serine proteasesenzymes that are encoded by a polynucleotide capable of hybridizing tothe polynucleotide encoding the protease of interest, under conditionsof medium to maximum stringency. For example, being equivalent meansthat an equivalent mature serine protease comprises at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, and/or at least about 99% sequenceidentity to the mature subtilisin protease having the amino acidsequence of SEQ ID NO:1.

The term “isolated” or “purified” refers to a material that is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, the material is said to be “purified”when it is present in a particular composition in a higher or lowerconcentration than exists in a naturally occurring or wild type organismor in combination with components not normally present upon expressionfrom a naturally occurring or wild type organism. For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. In some embodiments, such polynucleotides are partof a vector, and/or such polynucleotides or polypeptides are part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. In preferred embodiments, a nucleicacid or protein is said to be purified, for example, if it gives rise toessentially one band in an electrophoretic gel or blot.

The term “isolated”, when used in reference to a DNA sequence, refers toa DNA sequence that has been removed from its natural genetic milieu andis thus free of other extraneous or unwanted coding sequences, and is ina form suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (See e.g., Dynan and Tijan, Nature 316:774-78 [1985]).The term “an isolated DNA sequence” is alternatively referred to as “acloned DNA sequence”.

The term “isolated,” when used in reference to a protein, refers to aprotein that is found in a condition other than its native environment.In a preferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins. An isolated protein ismore than about 10% pure, preferably more than about 20% pure, and evenmore preferably more than about 30% pure, as determined by SDS-PAGE.Further aspects of the invention encompass the protein in a highlypurified form (i.e., more than about 40% pure, more than about 60% pure,more than about 80% pure, more than about 90% pure, more than about 95%pure, more than about 97% pure, and even more than about 99% pure), asdetermined by SDS-PAGE.

As used herein, the term, “combinatorial mutagenesis” refers to methodsin which libraries of variants of a starting sequence are generated. Inthese libraries, the variants contain one or several mutations chosenfrom a predefined set of mutations. In addition, the methods providemeans to introduce random mutations which were not members of thepredefined set of mutations. In some embodiments, the methods includethose set forth in U.S. patent application Ser. No. 09/699,250, filedOct. 26, 2000, hereby incorporated by reference. In alternativeembodiments, combinatorial mutagenesis methods encompass commerciallyavailable kits (e.g., QuikChange® Multisite, Stratagene, San Diego,Calif.).

As used herein, the term “library of mutants” refers to a population ofcells which are identical in most of their genome but include differenthomologues of one or more genes. Such libraries can be used, forexample, to identify genes or operons with improved traits.

As used herein, the term “starting gene” refers to a gene of interestthat encodes a protein of interest that is to be improved and/or changedusing the present invention.

As used herein, the term “multiple sequence alignment” (“MSA”) refers tothe sequences of multiple homologs of a starting gene that are alignedusing an algorithm (e.g., Clustal W).

As used herein, the terms “consensus sequence” and “canonical sequence”refer to an archetypical amino acid sequence against which all variantsof a particular protein or sequence of interest are compared. The termsalso refer to a sequence that sets forth the nucleotides that are mostoften present in a DNA sequence of interest. For each position of agene, the consensus sequence gives the amino acid that is most abundantin that position in the MSA.

As used herein, the term “consensus mutation” refers to a difference inthe sequence of a starting gene and a consensus sequence. Consensusmutations are identified by comparing the sequences of the starting geneand the consensus sequence resulting from an MSA. In some embodiments,consensus mutations are introduced into the starting gene such that itbecomes more similar to the consensus sequence. Consensus mutations alsoinclude amino acid changes that change an amino acid in a starting geneto an amino acid that is more frequently found in an MSA at thatposition relative to the frequency of that amino acid in the startinggene. Thus, the term consensus mutation comprises all single amino acidchanges that replace an amino acid of the starting gene with an aminoacid that is more abundant than the amino acid in the MSA.

As used herein, the term “enhanced combinatorial consensus mutagenesislibrary” refers to a CCM library that is designed and constructed basedon screening and/or sequencing results from an earlier round of CCMmutagenesis and screening. In some embodiments, the enhanced CCM libraryis based on the sequence of an initial hit resulting from an earlierround of CCM. In additional embodiments, the enhanced CCM is designedsuch that mutations that were frequently observed in initial hits fromearlier rounds of mutagenesis and screening are favored. In somepreferred embodiments, this is accomplished by omitting primers thatencode performance-reducing mutations or by increasing the concentrationof primers that encode performance-enhancing mutations relative to otherprimers that were used in earlier CCM libraries.

As used herein, the term “performance-reducing mutations” refer tomutations in the combinatorial consensus mutagenesis library that areless frequently found in hits resulting from screening as compared to anunscreened combinatorial consensus mutagenesis library. In preferredembodiments, the screening process removes and/or reduces the abundanceof variants that contain “performance-reducing mutations.”

As used herein, the term “functional assay” refers to an assay thatprovides an indication of a protein's activity. In particularlypreferred embodiments, the term refers to assay systems in which aprotein is analyzed for its ability to function in its usual capacity.For example, in the case of enzymes, a functional assay involvesdetermining the effectiveness of the enzyme in catalyzing a reaction.

As used herein, the term “target property” refers to the property of thestarting gene that is to be altered. It is not intended that the presentinvention be limited to any particular target property. However, in somepreferred embodiments, the target property is the stability of a geneproduct (e.g., resistance to denaturation, proteolysis or otherdegradative factors), while in other embodiments, the level ofproduction in a production host is altered. Indeed, it is contemplatedthat any property of a starting gene will find use in the presentinvention.

The term “property” or grammatical equivalents thereof in the context ofa nucleic acid, as used herein, refer to any characteristic or attributeof a nucleic acid that can be selected or detected. These propertiesinclude, but are not limited to, a property affecting binding to apolypeptide, a property conferred on a cell comprising a particularnucleic acid, a property affecting gene transcription (e.g., promoterstrength, promoter recognition, promoter regulation, enhancer function),a property affecting RNA processing (e.g., RNA splicing, RNA stability,RNA conformation, and post-transcriptional modification), a propertyaffecting translation (e.g., level, regulation, binding of mRNA toribosomal proteins, post-translational modification). For example, abinding site for a transcription factor, polymerase, regulatory factor,etc., of a nucleic acid may be altered to produce desiredcharacteristics or to identify undesirable characteristics.

The term “property” or grammatical equivalents thereof in the context ofa polypeptide (including proteins), as used herein, refer to anycharacteristic or attribute of a polypeptide that can be selected ordetected. These properties include, but are not limited to oxidativestability, substrate specificity, catalytic activity, thermal stability,alkaline stability, pH activity profile, resistance to proteolyticdegradation, K_(M), k_(cat), k_(cat)/k_(M) ratio, protein folding,inducing an immune response, ability to bind to a ligand, ability tobind to a receptor, ability to be secreted, ability to be displayed onthe surface of a cell, ability to oligomerize, ability to signal,ability to stimulate cell proliferation, ability to inhibit cellproliferation, ability to induce apoptosis, ability to be modified byphosphorylation or glycosylation, and/or ability to treat disease, etc.

The terms “modified sequence” and “modified genes” are usedinterchangeably herein to refer to a sequence that includes a deletion,insertion or interruption of naturally occurring nucleic acid sequence.In some preferred embodiments, the expression product of the modifiedsequence is a truncated protein (e.g., if the modification is a deletionor interruption of the sequence). In some particularly preferredembodiments, the truncated protein retains biological activity. Inalternative embodiments, the expression product of the modified sequenceis an elongated protein (e.g., modifications comprising an insertioninto the nucleic acid sequence). In some embodiments, an insertion leadsto a truncated protein (e.g., when the insertion results in theformation of a stop codon). Thus, an insertion may result in either atruncated protein or an elongated protein as an expression product.

As used herein, the terms “mutant sequence” and “mutant gene” are usedinterchangeably and refer to a sequence that has an alteration in atleast one codon occurring in a host cell's wild-type sequence. Theexpression product of the mutant sequence is a protein with an alteredamino acid sequence relative to the wild-type. The expression productmay have an altered functional capacity (e.g., enhanced enzymaticactivity).

The terms “mutagenic primer” or “mutagenic oligonucleotide” (usedinterchangeably herein) are intended to refer to oligonucleotidecompositions which correspond to a portion of the template sequence andwhich are capable of hybridizing thereto. With respect to mutagenicprimers, the primer will not precisely match the template nucleic acid,the mismatch or mismatches in the primer being used to introduce thedesired mutation into the nucleic acid library. As used herein,“non-mutagenic primer” or “non-mutagenic oligonucleotide” refers tooligonucleotide compositions which will match precisely to the templatenucleic acid. In one embodiment of the invention, only mutagenic primersare used. In another preferred embodiment of the invention, the primersare designed so that for at least one region at which a mutagenic primerhas been included, there is also non-mutagenic primer included in theoligonucleotide mixture. By adding a mixture of mutagenic primers andnon-mutagenic primers corresponding to at least one of the mutagenicprimers, it is possible to produce a resulting nucleic acid library inwhich a variety of combinatorial mutational patterns are presented. Forexample, if it is desired that some of the members of the mutant nucleicacid library retain their precursor sequence at certain positions whileother members are mutant at such sites, the non-mutagenic primersprovide the ability to obtain a specific level of non-mutant memberswithin the nucleic acid library for a given residue. The methods of theinvention employ mutagenic and non-mutagenic oligonucleotides which aregenerally between 10-50 bases in length, more preferably about 15-45bases in length. However, it may be necessary to use primers that areeither shorter than 10 bases or longer than 50 bases to obtain themutagenesis result desired. With respect to corresponding mutagenic andnon-mutagenic primers, it is not necessary that the correspondingoligonucleotides be of identical length, but only that there is overlapin the region corresponding to the mutation to be added. Primers may beadded in a pre-defined ratio according to the present invention. Forexample, if it is desired that the resulting library have a significantlevel of a certain specific mutation and a lesser amount of a differentmutation at the same or different site, by adjusting the amount ofprimer added, it is possible to produce the desired biased library.Alternatively, by adding lesser or greater amounts of non-mutagenicprimers, it is possible to adjust the frequency with which thecorresponding mutation(s) are produced in the mutant nucleic acidlibrary.

As used herein, the phrase “contiguous mutations” refers to mutationswhich are presented within the same oligonucleotide primer. For example,contiguous mutations may be adjacent or nearby each other, however, theywill be introduced into the resulting mutant template nucleic acids bythe same primer.

The terms “wild-type sequence,” or “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein engineering project. The wild-type sequence may encode eithera homologous or heterologous protein. A homologous protein is one thehost cell would produce without intervention. A heterologous protein isone that the host cell would not produce but for the intervention.Unless otherwise indicated, the amino acid position numbers refer tothose assigned to the mature Bacillus amyloliquefaciens subtilisinsequence of SEQ ID NO:1. The invention, however, is not limited to themutation of this particular subtilisin but extends to precursorproteases containing amino acid residues at positions which are“equivalent” to the particular identified residues in the Bacillusamyloliquefaciens subtilisin.

As used herein, the terms “protease variant,” “subtilisin variant,”“subtilisin protease variant,” are used in reference to proteases thatare similar to a wild-type subtilisin or a parent protease (e.g.,subtilisin) used as a starting point in protein engineering. Thesevariants are similar to the wild-type or other parent in their function,but have mutations in their amino acid sequence that make them differentin sequence from the wild-type or parent protease (e.g., subtilisin).

As used herein, the terms “modification” and “mutation” refers to anychange or alteration in an amino acid sequence. It is intended that theterm encompass substitutions, deletions, insertions, and/or replacementof amino acid side chains in an amino acid sequence of interest (e.g., asubtilisin sequence). It is also intended that the term encompasschemical modification of an amino acid sequence of interest (e.g., asubtilisin sequence).

The term “oxidation stable” refers to proteases of the present inventionthat retain a specified amount of enzymatic activity over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposed toor contacted with bleaching agents or oxidizing agents. In someembodiments, the proteases retain at least about 50%, about 60%, about70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%,about 96%, about 97%, about 98%, or about 99% proteolytic activity aftercontact with a bleaching or oxidizing agent over a given time period,for example, at least about 1 minute, about 3 minutes, about 5 minutes,about 8 minutes, about 12 minutes, about 16 minutes, about 20 minutes,etc. In some embodiments, the stability is measured as described in theExamples.

The term “chelator stable” refers to proteases of the present inventionthat retain a specified amount of enzymatic activity over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposed toor contacted with chelating agents. In some embodiments, the proteasesretain at least about 50%, about 60%, about 70%, about 75%, about 80%,about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about98%, or about 99% proteolytic activity after contact with a chelatingagent over a given time period, for example, at least about 10 minutes,about 20 minutes, about 40 minutes, about 60 minutes, about 100 minutes,etc. In some embodiments, the chelator stability is measured asdescribed in the Examples.

The terms “thermally stable” and “thermostable” refer to proteases ofthe present invention that retain a specified amount of enzymaticactivity after exposure to identified temperatures over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposedaltered temperatures. Altered temperatures include increased ordecreased temperatures. In some embodiments, the proteases retain atleast about 50%, about 60%, about 70%, about 75%, about 80%, about 85%,about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, orabout 99% proteolytic activity after exposure to altered temperaturesover a given time period, for example, at least about 60 minutes, about120 minutes, about 180 minutes, about 240 minutes, about 300 minutes,etc. In some embodiments, the thermostability is determined as describedin the Examples.

The term “enhanced stability” in the context of an oxidation, chelator,thermal and/or pH stable protease refers to a higher retainedproteolytic activity over time as compared to other serine proteases(e.g., subtilisin proteases) and/or wild-type enzymes.

The term “diminished stability” in the context of an oxidation,chelator, thermal and/or pH stable protease refers to a lower retainedproteolytic activity over time as compared to other serine proteases(e.g., subtilisin proteases) and/or wild-type enzymes.

The term “cleaning activity” refers to the cleaning performance achievedby the protease under conditions prevailing during the proteolytic,hydrolyzing, cleaning or other process of the invention. In someembodiments, cleaning performance is determined by the application ofvarious cleaning assays concerning enzyme sensitive stains, for examplegrass, blood, milk, or egg protein as determined by variouschromatographic, spectrophotometric or other quantitative methodologiesafter subjection of the stains to standard wash conditions. Exemplaryassays include, but are not limited to those described in WO 99/34011,and U.S. Pat. No. 6,605,458 (both of which are herein incorporated byreference), as well as those methods included in the Examples.

As used herein, “cleaning compositions” and “cleaning formulations”refer to compositions that find use in the removal of undesiredcompounds from items to be cleaned, such as fabric, dishes, contactlenses, other solid substrates, hair (shampoos), skin (soaps andcreams), teeth (mouthwashes, toothpastes) etc. The term encompasses anymaterials/compounds selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, gel,granule, or spray composition), as long as the composition is compatiblewith the perhydrolase and other enzyme(s) used in the composition. Thespecific selection of cleaning composition materials are readily made byconsidering the surface, item or fabric to be cleaned, and the desiredform of the composition for the cleaning conditions during use.

The terms further refer to any composition that is suited for cleaning,bleaching, disinfecting, and/or sterilizing any object and/or surface.It is intended that the terms include, but are not limited to detergentcompositions (e.g., liquid and/or solid laundry detergents and finefabric detergents; hard surface cleaning formulations, such as forglass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile and laundry pre-spotters, as well as dish detergents).

Indeed, the term “cleaning composition” as used herein, includes unlessotherwise indicated, granular or powder-form all-purpose or heavy-dutywashing agents, especially cleaning detergents; liquid, gel orpaste-form all-purpose washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car or carpet shampoos, bathroom cleaners; hair shampoos andhair-rinses; shower gels and foam baths and metal cleaners; as well ascleaning auxiliaries such as bleach additives and “stain-stick” orpre-treat types.

As used herein, the terms “detergent composition” and “detergentformulation” are used in reference to mixtures which are intended foruse in a wash medium for the cleaning of soiled objects. In somepreferred embodiments, the term is used in reference to launderingfabrics and/or garments (e.g., “laundry detergents”). In alternativeembodiments, the term refers to other detergents, such as those used toclean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is notintended that the present invention be limited to any particulardetergent formulation or composition. Indeed, it is intended that inaddition to perhydrolase, the term encompasses detergents that containsurfactants, transferase(s), hydrolytic enzymes, oxido reductases,builders, bleaching agents, bleach activators, bluing agents andfluorescent dyes, caking inhibitors, masking agents, enzyme activators,antioxidants, and solubilizers.

The term “cleaning effective amount” of a protease refers to thequantity of protease described hereinbefore that achieves a desiredlevel of enzymatic activity in a specific cleaning composition. Sucheffective amounts are readily ascertained by one of ordinary skill inthe art and are based on many factors, such as the particular proteaseused, the cleaning application, the specific composition of the cleaningcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, etc.

The term “cleaning adjunct materials,” as used herein, means any liquid,solid or gaseous material selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, granule,powder, bar, paste, spray, tablet, gel; or foam composition), whichmaterials are also preferably compatible with the protease enzyme usedin the composition. In some embodiments, granular compositions are in“compact” form, while in other embodiments, the liquid compositions arein a “concentrated” form.

The term “enhanced performance” in the context of cleaning activityrefers to an increased or greater cleaning activity of certain enzymesensitive stains such as egg, milk, grass or blood, as determined byusual evaluation after a standard wash cycle and/or multiple washcycles.

The term “diminished performance” in the context of cleaning activityrefers to an decreased or lesser cleaning activity of certain enzymesensitive stains such as egg, milk, grass or blood, as determined byusual evaluation after a standard wash cycle.

The term “comparative performance” in the context of cleaning activityrefers to at least about 60%, at least about 70%, at least about 80% atleast about 90%, or at least about 95% of the cleaning activity of acomparative subtilisin protease (e.g., commercially availableproteases), including but not limited to OPTIMASE™ protease (Genencor),PURAFECT™ protease products (Genencor), SAVINASE™ protease (Novozymes),BPN′-variants (See e.g., U.S. Pat. No. Re 34,606), RELASE™, DURAZYMET™,EVERLASE™, KANNASE™ protease (Novozymes), MAXACAL™, MAXAPEM™, PROPERASE™proteases (Genencor; See also, U.S. Pat. No. Re 34,606, and U.S. Pat.Nos. 5,700,676; 5,955,340; 6,312,936; and 6,482,628), and B. lentusvariant protease products (e.g., those described in WO 92/21760, WO95/23221 and/or WO 97/07770). Exemplary subtilisin protease variantsinclude, but are not limited to those having substitutions or deletionsat residue positions equivalent to positions 76, 101, 103, 104, 120,159, 167, 170, 194, 195, 217, 232, 235, 236, 245, 248, and/or 252 ofBPN′. Cleaning performance can be determined by comparing the proteasesof the present invention with those subtilisin proteases in variouscleaning assays concerning enzyme sensitive stains such as grass, bloodor milk as determined by usual spectrophotometric or analyticalmethodologies after standard wash cycle conditions.

As used herein, “fabric cleaning compositions” include hand and machinelaundry detergent compositions including laundry additive compositionsand compositions suitable for use in the soaking and/or pretreatment ofstained fabrics (e.g., clothes, linens, and other textile materials).

The “compact” form of the cleaning compositions herein is best reflectedby density and, in terms of composition, by the amount of inorganicfiller salt. Inorganic filler salts are conventional ingredients ofdetergent compositions in powder form. In conventional detergentcompositions, the filler salts are present in substantial amounts,typically about 17 to about 35% by weight of the total composition. Incontrast, in compact compositions, the filler salt is present in amountsnot exceeding about 15% of the total composition. In some embodiments,the filler salt is present in amounts that do not exceed about 10%, ormore preferably, about 5%, by weight of the composition. In someembodiments, the inorganic filler salts are selected from the alkali andalkaline-earth-metal salts of sulfates and chlorides. A preferred fillersalt is sodium sulfate.

As used herein, the term “surfactant” refers to any compound generallyrecognized in the art as having surface active qualities. Surfactantsgenerally include anionic, cationic, nonionic, and zwitterioniccompounds, which are further described, herein.

EXPERIMENTAL

The present invention is described in further detail in the followingExamples which are not in any way intended to limit the scope of theinvention as claimed. The following Examples are offered to illustrate,but not to limit the claimed invention.

In the experimental disclosure which follows, the followingabbreviations apply: PI (proteinase inhibitor), ppm (parts per million);M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol(moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm(grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters);ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm(millimeters); μm (micrometers); nm (nanometers); U (units); V (volts);MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) andhr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient);ND (not done); NA (not applicable); rpm (revolutions per minute); H₂O(water); dH₂O (deionized water); (HCl (hydrochloric acid); aa (aminoacid); by (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copyor complementary DNA); DNA (deoxyribonucleic acid); ssDNA (singlestranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl(sodium chloride); w/v (weight to volume); v/v (volume to volume); g(gravity); OD (optical density); Dulbecco's phosphate buffered solution(DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl,2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol,2.31 g/lKH₂PO₄, and 12.54 g/lK₂HPO₄); OD₂₈₀ (optical density at 280 nm);OD₆₀₀ (optical density at 600 nm); A₄₀₅ (absorbance at 405 nm); Vmax(the maximum initial velocity of an enzyme catalyzed reaction); PAGE(polyacrylamide gel electrophoresis); PBS (phosphate buffered saline[150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST (PBS+0.25%TWEEN® 20); PEG (polyethylene glycol); PCR (polymerase chain reaction);RT-PCR (reverse transcription PCR); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine(N-[tris-(hydroxymethyl)-methyl]glycine); CHES (2-(N-cyclo-hexylamino)ethane-sulfonic acid); TAPS(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS(3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide);DTT (1,4-dithio-DL-threitol); SA (sinapinic acid(s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic acid);Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP(Tris[2-carboxyethyl]phosphine); Ci (Curies); mCi (milliCuries); μCi(microCuries); HPLC (high pressure liquid chromatography); RP-HPLC(reverse phase high pressure liquid chromatography); TLC (thin layerchromatography); MALDI-TOF (matrix-assisted laserdesorption/ionization—time of flight); Ts (tosyl); Bn (benzyl); Ph(phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Therms aquaticus DNApolymerase); Klenow (DNA polymerase I large (Klenow) fragment); EGTA(ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid);EDTA (ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); HDL (high density liquid); MJ Research (MJResearch, Reno, Nev.); Baseclear (Baseclear BV, Inc., Leiden, theNetherlands); PerSeptive (PerSeptive Biosystems, Framingham, Mass.);ThermoFinnigan (ThermoFinnigan, San Jose, Calif.); Argo (ArgoBioAnalytica, Morris Plains, N.J.); Seitz EKS (SeitzSchenk FiltersystemsGmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, N.Y.);Spectrum (Spectrum Laboratories, Dominguez Rancho, Calif.); MolecularStructure (Molecular Structure Corp., Woodlands, Tex.); Accelrys(Accelrys, Inc., San Diego, Calif.); Chemical Computing (ChemicalComputing Corp., Montreal, Canada); New Brunswick (New BrunswickScientific, Co., Edison, N.J.); CFT (Center for Test Materials,Vlaardingen, the Netherlands); Test Fabrics (Test Fabrics, Inc., WestPittiston, Pa.), Procter & Gamble (Procter & Gamble, Inc., Cincinnati,Ohio); GE Healthcare (GE Healthcare, Chalfont St. Giles, UnitedKingdom); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (MegazymeInternational Ireland Ltd., Bray Business Park, Bray, Co., Wicklow,Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco,Wilmington, Del.); Corning (Corning Life Sciences, Corning, N.Y.); (NEN(NEN Life Science Products, Boston, Mass.); Pharma AS (Pharma AS, Oslo,Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis,Lewisville, Tex.); ATCC (American Type Culture Collection, Rockville,Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma ChemicalCo., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Piscataway, N.J.);NCBI (National Center for Biotechnology Information); Applied Biosystems(Applied Biosystems, Foster City, Calif.); BD Biosciences and/orClontech (BD Biosciences CLONTECH Laboratories, Palo Alto, Calif.);Operon Technologies (Operon Technologies, Inc., Alameda, Calif.); MWGBiotech (MWG Biotech, High Point, N.C.); Oligos Etc (Oligos Etc. Inc,Wilsonville, Oreg.); Bachem (Bachem Bioscience, Inc., King of Prussia,Pa.); Difco (Difco Laboratories, Detroit, Mich.); Mediatech (Mediatech,Herndon, Va.; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.); Oxoid (Oxoid Inc., Ogdensburg, N.Y.); Worthington (WorthingtonBiochemical Corp., Freehold, N.J.); GIBCO BRL or Gibco BRL (LifeTechnologies, Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica,Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (InvitrogenCorp., San Diego, Calif.); NEB (New England Biolabs, Ipswich, Mass.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (PierceBiotechnology, Rockford, Ill.); Takara (Takara Bio Inc. Otsu, Japan);Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science,Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Biodesign(Biodesign Intl., Saco, Me.); Aptagen (Aptagen, Inc., Herndon, Va.);Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville,N.C.); United States Testing (United States Testing Co., Hoboken, N.J.);Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.); R&DSystems (R&D Systems, Minneapolis, Minn.); Stratagene (StratageneCloning Systems, La Jolla, Calif.); Marsh (Marsh Biosciences, Rochester,N.Y.); Geneart (Geneart GmbH, Regensburg, Germany); DNA2.0 (DNA2.0,Menlo Park, Calif.); Gene Oracle (Gene Oracle, Mountain View, Calif.);Bio-Tek (Bio-Tek Instruments, Winooski, Vt.); Biacore (Biacore, Inc.,Piscataway, N.J.); PeproTech (PeproTech, Rocky Hill, N.J.); SynPep(SynPep, Dublin, Calif.); New Objective (New Objective brand; ScientificInstrument Services, Inc., Ringoes, N.J.); Waters (Waters, Inc.,Milford, Mass.); Matrix Science (Matrix Science, Boston, Mass.); Dionex(Dionex, Corp., Sunnyvale, Calif.); Monsanto (Monsanto Co., St. Louis,Mo.); Wintershall (Wintershall AG, Kassel, Germany); BASF (BASF Co.,Florham Park, N.J.); Huntsman (Huntsman Petrochemical Corp., Salt LakeCity, Utah); Enichem (Enichem Iberica, Barcelona, Spain); Fluka ChemieAG (Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades,Nev., Delft, the Netherlands); Dow Corning (Dow Corning Corp., Midland,Mich.); and Microsoft (Microsoft, Inc., Redmond, Wash.).

Example 1 Assays

In the following Examples, various assays were used as set forth belowfor ease in reading. Any deviations from the protocols provided beloware indicated in the Examples.

a. TCA Assay for Protein Content Determination in 96-Well MicrotiterPlates

For BPN′ (e.g., reference protease) and BPN′ variants, this assay wasstarted using filtered culture supernatant from microtiter plates grown3-4 days at 33° C. with shaking at 230 rpm and humidified aeration. Afresh 96-well flat bottom microtiter plate (MTP) was used for the assay.First, 100 μL/well of 0.25 N HCl was placed in each well. Then, 50 μL offiltered culture broth was added. The light scattering/absorbance at 405nm (use 5 sec mixing mode in the plate reader) was then determined, inorder to provide the “blank” reading. For the test, 100 μL/well of 15%(w/v) trichloroacetic acid (TCA) was placed in the plates and incubatedbetween 5 and 30 min at room temperature. The lightscattering/absorbance at 405 nm (use 5 sec mixing mode in the platereader) was then determined.

The equipment used was a Biomek FX Robot (Beckman Coulter) and aSpectraMAX (type 340; Molecular Devices) MTP Reader; the MTP's were fromCostar (type 9017). The equipment used was a Biomek FX Robot (BeckmanCoulter) and a SpectraMAX type 340 (Molecular Devices) MTP Reader; andthe MTPs were type 9017 (Costar).

The calculations were performed by subtracting the blank (no TCA) fromthe test reading with TCA to provide a relative measure of the proteincontent in the samples. If desired, a standard curve can be created bycalibrating the TCA readings with AAPF assays of clones with knownconversion factors. However, the TCA results are linear with respect toprotein concentration from 50 to 500 protein per ml (ppm) and can thusbe plotted directly against enzyme performance for the purpose ofchoosing good-performing variants. The turbidity/light scatter increasein the samples correlates to the total amount of precipitable protein inthe culture supernatant.

B. AAPF Protease Assay in 96-Well Microtiter Plates

In order to determine the protease activity of the proteases andvariants thereof of the present invention, the hydrolysis ofN-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide(suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mMTris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer);100 mM Tris buffer, pH 8.6, containing 10 mM CaCl₂ and 0.005% TWEEN®-80(Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stocksolution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer andmixed well for at least 10 seconds. The assay was performed by adding 10μl of diluted protease solution to each well, immediately followed bythe addition of 190 μl 1 mg/ml suc-AAPF-pNA working solution. Thesolutions were mixed for 5 sec., and the absorbance change in kineticmode (20 readings in 5 minutes) was read at 410 nm in an MTP reader, at25° C. The protease activity was expressed as AU (activity=ΔOD·min⁻¹ml⁻¹).

C. Surfactant and Chelant Stability Assays

LAS and LAS/EDTA stability was measured after incubation of the testprotease in the presence of LAS and LAS/EDTA respectively, as a functionof residual activity determined using the AAPF assay.

LAS Stability Method Reagents:

Dodecylbenzenesulfonate, Sodium salt (=LAS): Sigma D-2525

TWEEN®-80: Sigma P-8074

TRIS buffer (free acid): Sigma T-1378); 6.35 g is dissolved in about 960ml water; pH is adjusted to 8.2 with 4N HCl. Final concentration of TRISis 52.5 mM.LAS stock solution: Prepare a 10.5% LAS solution in MQ water (=10.5 gper 100 ml MQ)TRIS buffer-100 mM/pH 8.6 (100 mM Tris/0.005% Tween®-80)TRIS-Ca buffer, pH 8.6 (100 mM Tris/10 mM CaCl2/0.005% Tween-®80)

Hardware:

Flat bottom MTPs (Costar No. 9017)

Biomek FX ASYS Multipipettor Spectramax MTP Reader

iEMS Incubator/Shaker

Innova 4330 Incubator/Shaker

Biohit multichannel pipette

BMG Thermostar Shaker

A 0.063% LAS solution was prepared in 52.5 mM Tris buffer pH 8.2. Thesuc-AAPF-pNA working solution was prepared by adding 1 ml of 100 mg/mlsuc-AAPF-pNA stock solution (in DMSO) to 100 ml (100 mM) TRIS buffer, pH8.6. To dilute the supernatants, flat-bottomed plates were filled withdilution buffer and an aliquot of the supernatant was added and mixedwell. The dilution ratio depended on the concentration of the proteasecontrols in the growth plates (AAPF activity). The desired proteinconcentration was 80 ppm.

Ten μl of the diluted supernatant were added to 190 μl 0.063% LASbuffer/well. The MTP was covered with tape, shaken for a few seconds andplaced in an incubator (Innova 4230) at 45° C., for 60 minutes at 200rpm agitation. The initial activity (t=10 minutes) was determined after10 minutes of incubation by transferring 10 μl of the mixture in eachwell to a fresh MTP containing 190 μl suc-AAPF-pNA work solution. Thesesolutions were mixed well and the AAPF activity was measured using a MTPReader (20 readings in 5 minutes and 25° C.).

The final activity (t=60 minutes) was determined by removing another 10μl of solution from the incubating plate after 60 minutes of incubation.The AAPF activity was then determined as described above. The stabilityof the samples was determined by calculating the ration of the residualand initial AAPF activity as follows:

Residual Activity (%)=[t−60 value]*100/[t−10 value].

LAS/EDTA Stability Method

The stability of protease variants in the presence of a representativeanionic surfactant (LAS=linear alkylbene sulfonate, sodiumdodecylbenzenesulfonate-DOBS) and di-sodium EDTA was measured afterincubation under defined conditions and the residual activity wasdetermined using the AAPF assay. The reagents used were dodecyllbenzenesulfonate, sodium salt (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No.P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (SigmaNo. H-7523), unstress buffer: 50 mM HEPES (11.9 g/l)+0.005% TWEEN®-80,pH 8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l),10 mM EDTA (3.36 g/l), pH 8.0, reference protease and protease variantculture supernatants, containing 200-400 μg/ml protein. The equipmentused was V- or U-bottom MTP as dilution plates (Greiner 651101 and650161 respectively), F-bottom MTP (Corning 9017) for unstress andLAS/EDTA buffer as well as for suc-AAPF-pNA plates, Biomek FX (BeckmanCoulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMSIncubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), sealingtape: Nunc (236366)

The iEMS incubator/shaker (Thermo/Labsystems) was set at 29° C. Culturesupernatants were diluted into plates containing unstress buffer to aconcentration of 25 ppm (master dilution plate). 20 μl of sample fromthe master dilution plate was added to plates containing 180 μl unstressbuffer to give a final incubation concentration of 2.5 ppm. The contentswere mixed and kept at room temperature and a AAPF assay was performedon this plate. 20 μl of sample from the master dilution plate was alsoadded to plates containing 180 μl stress buffer (50 mM HEPES (11.9 g/l),0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0). The solutionswere mixed and immediately placed in 29° C. iEMS shaker for 30 min at400 rpm. Following 30 minutes of incubation, a AAPF assay was performedon the stress plate. The stability of the samples was determined bycalculating the ration of the residual and initial AAPF activity asfollows: Residual Activity (%)=[mOD·min−1 stressed]*100/[mOD·min−1unstressed].

D. Cleaning Performance Assays

The stain removal performance of the protease variants was determined incommercially available detergents. Heat inactivation of commercialdetergent formulas serves to destroy the enzymatic activity of anyprotein components while retaining the properties of non-enzymaticcomponents. Thus this method was suitable for preparing commerciallypurchased detergents for use in testing the enzyme variants of thepresent invention.

Microswatches:

Microswatches of ¼″ circular diameter were ordered and delivered by CFT(Vlaardingen, The Netherlands). Single microswatches or twomicroswatches were placed vertically in each well of a 96-well MTP toexpose the whole surface area (i.e., not flat on the bottom of thewell).

BMI Microswatch Assay

Microswatches containing blood milk and ink (BMI) of 0.25 inch circulardiameter were obtained from CFT. Before cutting of the swatches, thefabric (EMPA 116) was washed with water. One microswatch was verticallyplaced in each well of a 96-well microtiter plate in order to expose thewhole surface area (i.e., not flat on the bottom of the well). Thedesired detergent solution was prepared as described herein. Afterequilibrating the Thermomixer at 25° C., 190 μl of detergent solutionwas added to each well of the MTP, containing microswatches. To thismixture, 10 μl of the diluted enzyme solution was added so that thefinal enzyme concentration was 1 μg/ml (determined from BCA assay). TheMTP was sealed with tape and placed in the incubator for 30 minutes,with agitation at 1400 rpm. Following incubation under the appropriateconditions, 100 μl of the solution from each well was transferred into afresh MTP. The new MTP containing 100 μl of solution/well was read at405 nm using a MTP SpectraMax reader. Blank controls, as well as acontrol containing two microswatches and detergent but no enzyme werealso included.

“Pre-Washed” Swatch

This type of microswatch was pre-washed in deionised water for 20minutes at ambient temperature. After the pre-washing step, the swatcheswere put on top of paper towels to dry. The air-dried swatches were thenpunched using a ¼″ circular die on an expulsion press. Finally twomicroswatches were put into each well of a 96-well MTP vertically toexpose the whole surface area (i.e. not flat on the bottom of the well).

Detergents

For North American (NA) and Western European (WE) heavy duty liquidlaundry (HDL) detergents, heat inactivation was performed by placingpre-weighed liquid detergent (in a glass bottle) in a water bath at 95°C. for 2 hours. The detergents were purchased from local supermarketstores. Both un-heated and heated detergents were assayed within 5minutes of dissolving the detergent to accurately determine percentagedeactivated. Enzyme activity was tested by the AAPF assay.

For testing of enzyme activity in heat-inactivated detergents, workingsolutions of detergents were made from the heat inactivated stocks.Appropriate amounts of water hardness (6 gpg) and buffer were added tothe detergent solutions to match the desired conditions. The solutionswere mixed by vortexing or inverting the bottles.

Enzymes and Equipment

Samples of reference serine proteases variants thereof were obtainedfrom filtered culture broth of cultures grown in MTP plates. Theequipment used was a Biomek FX Robot (Beckman Coulter), a SpectraMAX MTPReader (type 340; Molecular Devices), an iEMS incubator/shaker(Thermo/Labsystems); F-bottom MTPs (Costar type 9017 used for readingreaction plates after incubation); and V-bottom MTPs (Greiner 651101used for pre-dilution of supernatant). In this assay, the proteaseshydrolyze the substrate and liberate pigment and insoluble particlesfrom the substrate. Thus the rate of turbidity is a measure of enzymeactivity.

The stain removal performance of reference serine proteases and variantstherefrom on microswatches was determined on a MTP scale in commerciallyavailable heat-inactivated detergent. The reagents used were: 5 mMHEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer, 3:1 Ca:Mg for medium waterhardness. (CaCl₂: MgCl2.6H2O); 15000 grains per gallon (gpg) stockdiluted to 6 gpg, 2 BMI (blood/milk/ink) swatches per plate: EMPA-116BMI cotton swatches processed by CFT: pre-rinsed and punched 2 swatchesper well, and heat inactivated TIDE® 2× Cold off-the-shelf detergent inwhich lack of protease activity was confirmed.

TABLE 1-2 Working Detergent Solutions Temp Detergent Detergent (C.) g/LpH Buffer gpg Protease TIDE ® 2X Cold 16 0.98 8 5 mM 6 BPN′ HEPES TIDE ®2X Cold 32 0.98 8 5 mM 6 BPN′ HEPES TIDE ® 2X Cold 16 0.98 7 5 mM 6 BPN′MOPS

The incubator was set at the desired temperature (16° C. or 32° C.). 10μL samples from the master dilution plate of ˜10 ppm enzyme was added toBMI 2-swatch plates with 190 μL working detergent solutions listedabove. The volume was adjusted to give final concentration of 0.5 ppmfor variants in the assay plates. The plates were immediatelytransferred to iEMS incubators and incubated for 30 minutes with 1400rpm shaking at given temperature. Following incubation, 100 μL ofsupernatant was transferred into a new 96-well plate and the absorbancewas measured in MTP Reader at 405 nm and/or 600 nm. Control wells,containing 1 or 2 microswatches and detergent without the addition ofprotease samples were also included in the test. The measurement at 405nm provides a higher value and tracks pigment removal, while themeasurement at 600 nm tracks turbidity and cleaning.

Calculation of the Stain Removal Activity for all Microswatch AssayMethods:

The absorbance value obtained was corrected for the blank value(substrate without enzyme), providing a measure of hydrolytic activity.For each sample (variant) the performance index was calculated. Theperformance index compares the performance of the variant (actual value)and the standard enzyme (theoretical value) at the same proteinconcentration. In addition, the theoretical values can be calculated,using the parameters of the Langmuir equation of the standard enzyme.

E. Relative Specific Activity of Proteases and Variants Thereof

In order to discriminate the protease variants, the relative specificactivity was calculated using suc-AAPF-pNA as a substrate, which enabledthe comparison and ranking of the variants versus the wild-type orstandard protease. The specific activity on the suc-AAPF-pNA substratewas determined by dividing the proteolytic activity by the measuredTCA-values of each sample, using the assays described above. Using thesevalues, the relative specific activity was calculated (specific activityof variant/specific activity of reference protease).

F. Performance Index

The performance index compares the performance of the variant (actualvalue) and the standard or reference protease (theoretical value) at thesame protein concentration. In addition, the theoretical values can becalculated, using the parameters of the binding curve (i.e., Langmuirequation) of the standard protease. A performance index (PI) that isgreater than 1 (PI>1) identifies a better variant as compared to thestandard (e.g., wild-type), while a PI of 1 (PI=1) identifies a variantthat performs the same as the standard, and a PI that is less than 1(PI<1) identifies a variant that performs worse than the standard. Thus,the PI identifies winners, as well as variants that are less desirablefor use under certain circumstances.

Example 2 Stain Removal Performance of BPN′ Multiple Mutation Library(MML) Variants

BPN′ multiple mutation libraries (or combinatorial libraries) wereproduced by Geneart or DNA 2.0, using BPN′ as the parent protein.Protein concentration of culture supernatants was determined by TCAprecipitation as described in Example 1. The stain removal performanceof the variants was tested in laundry applications on EMPA 116 swatches(BMI stain, CFT) at pH 8/16° C., pH 7/16° C. and pH 8/32° C. usingmethods described in Example 1, with the following modifications. Thetest detergent used was heat inactivated TIDE® 2× Cold detergent(Procter & Gamble), prepared as described in Example 1. Heatinactivation of commercial detergent formulas serves to destroy theendogenous enzymatic activity of any protein components while retainingthe properties of non0enzymatic components.

Heat inactivation of the detergents was performed by placing pre-weighedamounts of liquid detergent (in a glass bottle) in a water bath at 95°C. for 2 hours. The detergent was purchased from local supermarketstores. Both unheated and heated detergents were assayed within 5minutes of dissolving the detergent, in order to accurately determinepercentage deactivated. Enzyme activity was tested by AAPF assay.Functionality of BPN′ variants was quantified as a performance index(Pi) (i.e., the ratio of performance of a variant relative to parentBPN′). Results are shown in Table 2-1. BPN′ variants showing a Pi valuegreater than or equal to 0.5 for one or more BMI stain removalperformance tests and/or TCA precipitation showed improved cleaningbenefits and/or expression. Performance indices less than or equal to0.05 were fixed to 0.05 and indicated in bold italics in the table. Forevery variant with a TCA protein performance index less than or equal to0.05, all values were fixed at 0.05.

TABLE 2-1 P_(i) Values of BPN' Variants Tested for Expression (TCA) andStain Removal Performance (BMI pH 8/16° C., BMI pH 7/16° C., and BMI pH8/32° C.) BMI BMI BMI Variant Code TCA pH 8/16° C. pH 7/16° C. pH 8/32°C. Parent BPN' 1.00 1.00 1.00 1.00 FNA (BPN'Y217L) 1.01 1.12 1.08 1.08G97A-G128A-Y217Q 1.33 1.4 1.24 1.23 G97A-L126A-G128A 0.56 0.83 0.85 0.89G97A-L126A-G128A-Y217Q 0.45 0.57 0.79 0.79 G97A-L126A-Y217Q 1.11 1.321.24 1.23 G97A-M124V-G128A 0.63 1.16 1.1 1.07 G97A-M124V-G128A-Y217Q0.52 1.12 1.04 1.06 G97A-M124V-L126A 1.63 1.29 1.21 1.18G97A-M124V-L126A-G128A 0.66 1.04 1.15 0.95 G97A-M124V-L126A-Y217Q 1.791.35 1.22 1.16 G97A-M124V-Y217Q 0.72 0.05 0.05 0.05 G97A-N123G-G128A 0.50.54 0.55 0.55 G97A-N123G-G128A-Y217Q 0.36 0.5 0.59 0.53G97A-N123G-L126A 0.64 0.46 0.4 0.65 G97A-N123G-L126A-G128A 0.47 0.050.12 0.05 G97A-N123G-L126A-Y217Q 0.62 0.24 0.38 0.43 G97A-N123G-M124V0.38 0.38 0.34 0.53 G97A-N123G-M124V-G128A 0.36 0.05 0.05 0.05G97A-N123G-M124V-L126A 0.58 0.4 0.4 0.49 G97A-N123G-M124V-Y217Q 0.050.05 0.05 0.05 G97A-N123G-Y217Q 0.55 1.35 1.24 1.13 L126A-G128A-Y217Q0.56 0.8 0.84 0.79 L96T-G128A-Y217Q 0.3 0.28 0.32 0.58 L96T-G97A-G128A0.39 0.41 0.3 0.54 L96T-G97A-G128A-Y217Q 0.5 0.21 0.34 0.5L96T-G97A-L126A 0.47 0.16 0.1 0.32 L96T-G97A-L126A-G128A 0.43 0.1 0.190.09 L96T-G97A-L126A-Y217Q 0.59 0.05 0.11 0.05 L96T-G97A-M124V 0.49 1.171.03 1.02 L96T-G97A-M124V-G128A 0.54 0.19 0.31 0.32L96T-G97A-M124V-L126A 0.88 0.34 0.52 0.57 L96T-G97A-M124V-Y217Q 0.431.08 1.06 0.95 L96T-G97A-N123G 0.35 0.13 0.09 0.21 L96T-G97A-N123G-G128A0.44 0.05 0.06 0.05 L96T-G97A-N123G-L126A 0.48 0.05 0.05 0.05L96T-G97A-N123G-M124V 0.49 0.22 0.28 0.24 L96T-G97A-N123G-Y217Q 0.490.05 0.22 0.07 L96T-G97A-Y217Q 0.46 1.3 1.11 1.08 L96T-L126A-G128A 0.560.05 0.05 0.05 L96T-L126A-G128A-Y217Q 0.42 0.05 0.06 0.05L96T-L126A-Y217Q 0.51 0.11 0.06 0.36 L96T-M124V-G128A 0.49 0.59 0.530.67 L96T-M124V-G128A-Y217Q 0.42 0.34 0.54 0.51 L96T-M124V-L126A 0.680.62 0.6 0.79 L96T-M124V-L126A-G128A 0.48 0.05 0.08 0.09L96T-M124V-L126A-Y217Q 0.73 0.43 0.53 0.67 L96T-M124V-Y217Q 0.51 1.231.06 1.03 L96T-N123G-G128A 0.48 0.05 0.05 0.05 L96T-N123G-G128A-Y217Q0.49 0.05 0.09 0.08 L96T-N123G-L126A 0.43 0.05 0.05 0.05L96T-N123G-L126A-G128A 0.48 0.05 0.05 0.05 L96T-N123G-L126A-Y217Q 0.350.05 0.05 0.05 L96T-N123G-M124V 0.51 0.05 0.05 0.07L96T-N123G-M124V-G128A 0.41 0.05 0.06 0.05 L96T-N123G-M124V-L126A 0.420.06 0.14 0.05 L96T-N123G-M124V-Y217Q 0.48 0.06 0.12 0.14L96T-N123G-Y217Q 0.45 0.15 0.05 0.27 M124V-G128A-Y217Q 0.77 1.27 1.181.03 M124V-L126A-G128A 0.72 1.12 1.1 1.02 M124V-L126A-G128A-Y217Q 0.521.21 1.22 1.12 M124V-L126A-Y217Q 1.71 1.39 1.16 1.17 N123G-G128A-Y217Q0.13 1 1.25 1.08 N123G-L126A-G128A 0.49 0.13 0.17 0.33N123G-L126A-G128A-Y217Q 0.39 0.13 0.11 0.21 N123G-L126A-Y217Q 0.91 0.370.43 0.55 N123G-M124V-G128A 0.6 0.05 0.13 0.38 N123G-M124V-G128A-Y217Q0.63 0.05 0.05 0.05 N123G-M124V-L126A 0.81 0.48 0.44 0.62N123G-M124V-L126A-G128A 0.46 0.24 0.47 0.44 N123G-M124V-L126A-Y217Q 0.710.35 0.44 0.48 N123G-M124V-Y217Q 0.6 0.35 0.42 0.51 N62Q-G128A-Y217Q0.69 1.3 1.2 1.13 N62Q-G97A-G128A 0.69 1.15 1.14 1.07N62Q-G97A-G128A-Y217Q 0.67 1.15 1.17 0.98 N62Q-G97A-L126A 0.94 0.74 0.740.96 N62Q-G97A-L126A-G128A 0.47 0.05 0.23 0.28 N62Q-G97A-L126A-Y217Q0.87 0.58 0.77 0.72 N62Q-G97A-M124V 1.17 1.19 1.08 1.11N62Q-G97A-M124V-G128A 0.57 0.52 0.62 0.64 N62Q-G97A-M124V-L126A 1.211.03 1.08 1.03 N62Q-G97A-M124V-Y217Q 0.94 1.21 1.14 1.04 N62Q-G97A-N123G0.37 0.58 0.56 0.58 N62Q-G97A-N123G-G128A 0.44 0.05 0.08 0.09N62Q-G97A-N123G-L126A 0.53 0.05 0.07 0.06 N62Q-G97A-N123G-M124V 0.370.05 0.07 0.05 N62Q-G97A-N123G-Y217Q 0.51 0.72 0.8 0.72 N62Q-G97A-Y217Q1.72 1.38 1.18 1.32 N62Q-L126A-G128A 0.48 0.12 0.13 0.36N62Q-L126A-G128A-Y217Q 0.47 0.08 0.27 0.15 N62Q-L126A-Y217Q 0.99 0.930.95 1 N62Q-L96T-G128A 0.48 0.2 0.1 0.31 N62Q-L96T-G128A-Y217Q 0.9 0.110.26 0.27 N62Q-L96T-G97A 0.48 0.88 0.82 0.9 N62Q-L96T-G97A-G128A 0.530.15 0.2 0.25 N62Q-L96T-G97A-L126A 0.58 0.05 0.16 0.14N62Q-L96T-G97A-M124V 0.64 0.68 0.74 0.72 N62Q-L96T-G97A-N123G 0.37 0.090.25 0.14 N62Q-L96T-G97A-Y217Q 0.57 0.85 0.74 0.83 N62Q-L96T-L126A 0.50.05 0.05 0.05 N62Q-L96T-L126A-G128A 0.51 0.09 0.2 0.18N62Q-L96T-L126A-Y217Q 0.43 0.05 0.19 0.23 N62Q-L96T-M124V 0.44 0.78 0.720.79 N62Q-L96T-M124V-G128A 0.74 0.05 0.14 0.11 N62Q-L96T-M124V-L126A 0.70.25 0.3 0.44 N62Q-L96T-M124V-Y217Q 0.55 0.73 0.78 0.76 N62Q-L96T-N123G0.36 0.05 0.05 0.06 N62Q-L96T-N123G-G128A 0.44 0.05 0.05 0.05N62Q-L96T-N123G-L126A 0.42 0.05 0.14 0.18 N62Q-L96T-N123G-M124V 0.410.05 0.05 0.05 N62Q-L96T-N123G-Y217Q 0.5 0.05 0.08 0.05 N62Q-L96T-Y217Q0.58 0.86 0.64 0.85 N62Q-M124V-G128A 0.46 0.75 0.71 0.71N62Q-M124V-G128A-Y217Q 0.45 0.43 0.59 0.46 N62Q-M124V-L126A 1.62 1.21.06 1.09 N62Q-M124V-L126A-G128A 0.51 0.23 0.45 0.42N62Q-M124V-L126A-Y217Q 0.97 0.05 0.09 0.05 N62Q-M124V-Y217Q 1.04 1.221.09 0.89 N62Q-N123G-G128A 0.41 0.05 0.05 0.06 N62Q-N123G-G128A-Y217Q0.43 0.05 0.13 0.09 N62Q-N123G-L126A 0.55 0.05 0.05 0.14N62Q-N123G-L126A-G128A 0.5 0.05 0.05 0.05 N62Q-N123G-L126A-Y217Q 0.540.05 0.09 0.05 N62Q-N123G-M124V 0.48 0.05 0.05 0.05N62Q-N123G-M124V-G128A 0.45 0.05 0.05 0.05 N62Q-N123G-M124V-L126A 0.340.05 0.11 0.07 N62Q-N123G-M124V-Y217Q 0.41 0.05 0.08 0.1N62Q-N123G-Y217Q 0.49 0.97 0.8 0.92

Example 3 Saturation Libraries at 97-128-217 and Additional Mutations

Saturation libraries at positions 97-128-217 in BPN′ (parent) wereproduced by DNA 2.0. Protein concentration of culture supernatants wasdetermined by TCA precipitation as described in Example 1. The stainremoval performance of the variants was tested in laundry applicationson EMPA 116 swatches (BMI stain, CFT) at pH8/16° C. using methodsdescribed in Example 1, as described in Example 2. Enzyme activity wastested by AAPF assay as described in Example 1. Functionality of BPN′variants was quantified as a performance index (Pi) (i.e., the ratio ofperformance of a variant relative to FNA). Results are shown in Table3-1. BPN′ variants showing a Pi value greater than or equal to 0.5 forBMI stain removal performance test and/or TCA precipitation showedimproved cleaning benefits and/or expression.

TABLE 3-1 P_(i) Values of BPN' Variants Tested for Protein Determination(TCA) and Stain Removal Performance (BMI pH 8/16° C.) Variant NumberVariants TCA BMI pH 8 16° C. 1 G97N-G128A-Y217M 1.09 1.43 2G97G-G128S-Y217E 1.53 1.39 3 G97A-G128A-Y217Q 1.34 1.36 4G97M-G128S-Y217E 1.20 1.35 5 G97A-G128S-Y217Q 1.90 1.33 6G97D-G128S-Y217Q 1.55 1.33 7 G97M-G128G-Y217M 1.61 1.33 8G97G-G128S-Y217Q 1.64 1.32 9 G97S-G128S-Y217Q 1.52 1.32 10G97G-G128A-Y217Q 1.33 1.30 11 G97S-G128A-Y217E 1.03 1.30 12G97A-G128S-Y217L 2.18 1.29 13 G97A-G128A-Y217N 1.22 1.28 14G97Q-G128S-Y217L 1.89 1.28 15 G97A-G128A-Y217M 1.45 1.28 16G97A-G128A-Y217S 1.35 1.27 17 G97D-G128A-Y217Q 1.14 1.27 18G97M-G128S-Y217Q 0.99 1.27 19 G97Q-G128G-Y217D-S87Y 1.45 1.27 20G97S-G128A-Y217N 1.09 1.27 21 G97A-G128S-Y217T 1.61 1.27 22G97D-G128S-Y217E 1.01 1.27 23 G97D-G128A-Y217L 1.38 1.26 24G97G-G128S-Y217E-S78P- 1.00 1.26 A272T 25 G97T-G128S-Y217D 1.13 1.26 26G97D-G128A-Y2171 0.99 1.26 27 G97Q-G128S-Y217Q 1.59 1.26 28G97G-G128A-Y217D 1.12 1.25 29 G97Q-G128A-Y217N 1.09 1.25 30G97S-G128A-Y217M 1.41 1.25 31 G97S-G128S-Y217N 1.55 1.25 32G97S-G128S-Y217M 1.53 1.25 33 G97E-G128S-Y217M 1.58 1.24 34G97S-G128P-Y217Q 0.99 1.24 35 G97T-G128S-Y217Q 1.06 1.24 36G97D-G128S-Y217Q-A73T 1.18 1.23 37 G97E-G128S-Y217N 1.24 1.23 38G97G-G128A-Y2171 1.51 1.23 39 G97Q-G128A-Y217D 1.14 1.23 40G97Q-G128S-Y217M 1.98 1.23 41 G97R-G128T-Y217Q-S162P 0.68 1.23 42G97S-G128S-Y217D 1.50 1.23 43 G97T-G128P-Y2171 1.30 1.23 44G97Q-G128G-Y217E 1.58 1.23 45 G97C-G128G-Y217N 1.26 1.22 46G97D-G128S-Y217H 1.49 1.22 47 G97M-G128S-Y217L 1.02 1.22 48G97M-G128S-Y217N 0.98 1.21 49 G97S-G128S-Y217E 0.59 1.21 50G97M-G128S-Y2171 1.11 1.19 51 G97A-G128P-Y217A 0.84 1.18 52G97R-G128S-Y217D 0.95 1.16 53 G97D-G128A-Y217D 0.75 1.15 54G97V-G128G-Y217D 0.71 1.14 55 G97V-G128G-Y217E 0.72 1.13 56G97A-G128G-Y217T 1.29 1.12 57 G97G-G128N-Y217L 0.82 1.12 58G97D-G128A-Y217T 0.69 1.11 59 G97M-G128A-Y217E 0.64 1.11 60G97M-G128A-Y217N 0.58 1.06 FNA G97G-G128G-Y217L 1.00 1.00

Example 4 Additional Library Designs and Stain Removal Performance ofVariants

Additional BPN′ multiple mutation libraries were produced by Geneart orGene Oracle, using BPN′: G97A-G128A-Y217Q protein as the parentmolecule. Results of experiments conducted to determine stain removalactivity (microswatch assay to determine stain removal performance inlaundry applications using EMPA 116 swatches (BMI stain, CFTVlaardingen) (BMI pH8, BMI pH7, BMI 32° C.), protein determination byTCA precipitation, and LAS/EDTA stability (tests of properties ofinterest) of BPN′ variants are shown in Tables 4-1, 4-2, 4-3, and 4-4.

The results were obtained using the methods described in Example 1, withthe following modifications for the stain removal performance assay. Thetest detergent used was heat inactivated TIDE® 2× Cold detergent(Procter & Gamble, Cincinnati, Ohio, USA).

Heat inactivation of commercial detergent formulas serves to destroy theendogenous enzymatic activity of any protein components while retainingthe properties of nonenzymatic components. Heat inactivation of thedetergents was performed by placing pre-weighed amounts of liquiddetergent (in a glass bottle) in a water bath at 95° C. for 2 hours. Thedetergent was purchased from local supermarket stores. Both unheated andheated detergents were assayed within 5 minutes of dissolving thedetergent to accurately determine percentage deactivated. Enzymeactivity was tested by AAPF assay. As described throughout herein,functionality of BPN′ variants was quantified as a performance index(Pi), which is the ratio of performance of a variant to parent proteinBPN′: G97A-G128A-Y217Q. BPN′: G97A-G128A-Y217Q variants showing a Pivalue greater or equal than 0.5 for BMI stain removal performance and/orTCA precipitation showed improved cleaning benefits and/or expression.Performance indices less than or equal to 0.05 were fixed to 0.05 andindicated in bold italics in the table.

TABLE 4-1 Stain removal performance of multiple mutation variants ofBPN': G97A-G128A-Y217Q Parent BMI BMI BMI Variant pH 8 pH 7 32° C. TCAS145D 1.06 1.03 1.18 0.96 P239R 1.03 0.98 1.11 0.67 N61E P129E S162KK213L N240K 1.02 0.93 1.05 0.98 N61E 1.02 0.98 1.09 1.33 P40E A144KK213L 1.01 0.92 0.91 0.80 P129E 0.99 1.01 1.03 1.04 N61E P129E S159K0.99 0.98 1.07 1.19 K213L 0.98 1.00 1.03 1.06 S87D 0.98 1.03 1.02 0.75Q206E 0.97 0.96 0.98 1.01 S24R P40E S145D S159K K213L 0.97 0.96 0.980.91 K265N 0.96 1.00 1.00 0.93 S24R 0.96 0.87 0.99 0.99 P40E 0.96 0.960.96 0.80 Q275E 0.95 1.01 0.95 0.99 P129E S145D N240K 0.95 0.97 0.980.82 A144K 0.95 0.87 0.95 0.93 S159K 0.94 0.90 0.96 1.03 S162K 0.94 0.921.01 0.96 N240K 0.94 0.92 1.03 0.71 S24R S87D Q206E 0.90 0.95 0.98 0.83S87D S162K K265N 0.89 0.96 0.93 0.64 N61E S145D S162K K213L T242R 0.880.95 0.89 0.72 S87D A144K S145D S159K Q275E 0.88 0.91 0.96 0.72 S24RS87D A144K K265N Q275E 0.88 0.94 0.93 0.55 T242R 0.87 0.84 0.83 0.53P40E N61E P129E A144K S162K K213L N240K 0.85 0.88 0.89 0.64 S24R P40EN61E A144K Q206E K213L T242R 0.85 0.81 0.91 0.45 P129E P239R K265N 0.830.93 0.86 0.59 S24R P129E Q206E N240K K265N 0.81 0.86 0.82 0.63 P40ES145D S159K S162K K213L P239R 0.78 0.82 0.83 0.47 Q275E Q103E 0.67 0.840.72 0.46 S87D T242R Q275E 0.60 0.74 0.62 0.38 P129E S145D N240K T242RK265N 0.58 0.72 0.68 0.37 N62R 0.58 0.63 0.64 0.44 P40E N61E S87D S162KT242R 0.55 0.62 0.58 0.29 P40E N61E S87D P129E S159K S162K T242R 0.520.47 0.57 0.27 P40E N61E Q103E A144K S159K S162K Q275E 0.44 0.50 0.530.30 N61E Q103E N240K 0.44 0.62 0.51 0.42 S87D T242R K265N 0.42 0.600.48 0.28 Q103E S162K Q206E K213L P239R 0.42 0.53 0.46 0.31 P40E N61EQ103E S159K S162K K213L P239R 0.39 0.43 0.50 0.25 P40E N61E Q103E S159KS162K K213L N240K 0.38 0.40 0.49 0.26 N61E Q103E A144K K213L T242R 0.360.38 0.49 0.27 N62R K265N Q275E 0.29 0.42 0.32 0.38 N62R S159K Q206EK265N Q275E 0.26 0.35 0.33 0.35 S24R Q103E P129E N240K K265N 0.24 0.290.27 0.24 N62R S87D P129E S145D S159K S162K Q275E 0.24 0.25 0.33 0.30Q103E P129E T242R 0.23 0.28 0.31 0.28 S24R N61E Q103E P129E K213L N240KT242R 0.22 0.15 0.27 0.25 P40E N62R S145D S159K S162K Q206E Q275E 0.210.24 0.31 0.32 S24R P40E N61E S87D Q103E S159K S162K 0.19 0.21 0.25 0.23K213L N240K N62R S87D S145D S159K S162K K265N Q275E 0.19 0.22 0.19 0.33P40E N61E N62R S87D S159K S162K K265N 0.18 0.19 0.21 0.28 N61E S87DQ103E S159K S162K K213L T242R 0.17 0.13 0.17 0.25 N61E Q103E P129EP239RN240K 0.14 0.18 0.23 0.33 P40E N62R S87D S145D S159K S162K Q275E0.14 0.14 0.27 0.33 S24RN62R S87D S145D K265N 0.13 0.22 0.16 0.32 N62RS87D S145D S159K S162K K213L N240K 0.11 0.11 0.12 0.32 K265N Q275E S24RN61E Q103E P129E Q206E P239R N240K 0.09 0.05 0.05 0.26 N61E Q103E A144KQ206E K213L N240K 0.07 0.05 0.15 0.31 T242R S24R Q103E P129E S145DP239RN240K 0.06 0.05 0.05 0.26 K265N S24R N61E Q103E P129E Q206E K213LP239R 0.05 0.05 0.05 0.43 N240K T242R N61E Q103E P129E K213L P239RN240K0.05 0.05 0.05 0.38 T242R P40E N62R S87D Q103E A144K S159K Q275E 0.050.05 0.05 0.25 N61E N62R S87D Q103E S159K S162K K213L 0.05 0.05 0.050.33 T242R Q275E S24R P40E N61E Q103E P129E A144K K213L 0.05 0.05 0.050.36 P239R N240K T242R K265N S24R Q103E P129E S145D Q206E P239R 0.050.05 0.05 0.38 N240K T242R K265N P40E N62R S87D Q103E S162K 0.05 0.050.05 0.29 N61E Q103E Q206E K213L P239RN240K 0.05 0.05 0.05 0.32 T242RS24R P40E N61E Q103E P129E A144K Q206E 0.05 0.05 0.05 0.37 K213LP239RN240K T242R S24R N61E Q103E P129E A144K S145D Q206E 0.05 0.05 0.050.38 K213L P239RN240K T242R S24R N61E Q103E P129E S145D P239RN240K 0.050.05 0.05 0.35 T242R K265N S24R P40E N61E Q103E P129E A144K S145D 0.050.05 0.05 0.34 K213L P239RN240K T242R N61E S87D Q103E P129E S159K S162KK213L 0.05 0.05 0.05 0.25 N240K T242R S24R P40E N61E S87D Q103E P129EA144K 0.05 0.05 0.05 0.35 S162K Q206E K213L P239RN240K T242R S24R N61ES87D Q103E P129E A144K Q206E 0.05 0.05 0.05 0.34 K213L P239RN240K T242RS24RN62R P129E S145D P239R K265N Q275E 0.05 0.05 0.05 0.28 P40E N61ES87D Q103E S145D S159K S162K 0.05 0.05 0.05 0.30 K213L P239RN240K T242RP40E N61E Q103E P129E A144K S162K Q206E 0.05 0.05 0.05 0.37 K213L P239RN240K T242R S24RN62R P129E Q206E N240K K265N Q275E 0.05 0.05 0.05 0.28P40E N62R S87D S145D S159K S162K N240K 0.05 0.05 0.05 0.27 K265N Q275EP40E N62R S87D S159K S162K K265N Q275E 0.05 0.06 0.06 0.30 S24R P40EN61E Q103E P129E S162K Q206E 0.05 0.05 0.05 0.37 K213L P239R N240K T242RN61E Q103E P129E A144K Q206E K213L 0.05 0.05 0.05 0.34 P239R N240K T242RS24R P40E N61E S87D Q103E P129E A144K 0.05 0.05 0.05 0.33 K213L P239RN240K T242R P40E N61E Q103E P129E A144K K213L P239R 0.05 0.05 0.05 0.34N240K T242R S24R N61E Q103E P129E A144K Q206E K213L 0.05 0.05 0.05 0.43P239RN240K T242R K265N

The LAS/EDTA stability of the BPN′ multiple mutation libraries wastested and compared to BPN′: G97A-G128A-Y217Q. The LAS/EDTA assay wasperformed as described in LAS Stability assay in Example 1 except thatthe stress buffer contained 0.1% LAS+10 mM EDTA and the stress plateswere incubated at 40° C. for 30 minutes. The functionality of BPN′variants was quantified as a performance index (Pi), which is the ratioof performance of a variant to parent protein BPN′: G97A-G128A-Y217Q.The results are shown in Table 4-2.

TABLE 4-2 Stain removal performance of multiple mutation variants ofBPN′: G97A-G128A-Y217Q Parent Variant Mutations BMI pH8 BMI pH7 TCALAS/EDTA LHS1 S53G 1.05 1.03 1.18 0.98 LHS2 F58G 0.38 0.43 0.50 0.99LHS3 S78N 0.99 1.00 1.00 1.50 LHS4 Y104N 0.36 0.53 0.73 1.49 LHS5 I111V0.91 0.95 0.75 1.19 LHS6 A114G 0.71 0.72 0.60 0.93 LHS7 N117S 0.80 0.880.66 0.97 LHS8 S125A 0.73 0.79 0.85 0.97 LHS9 S132N 0.86 0.80 0.59 1.03LHS10 P239V 0.42 0.43 0.47 1.22 LHS11 S53G F58G 0.67 0.71 0.49 0.75LHS12 S53G S78N 0.98 0.99 1.10 1.44 LHS13 S53G Y104N 0.35 0.56 0.71 1.47LHS14 S53G I111V 0.99 0.99 0.83 1.20 LHS15 S53G A114G 0.85 0.91 0.641.15 LHS16 S53G N117S 0.93 1.00 0.75 1.30 LHS17 S53G S125A 0.71 0.760.85 0.87 LHS18 S53G S132N 0.93 0.93 0.74 1.03 LHS19 S53G P239V 0.520.59 0.46 1.12 LHS20 F58G S78N 0.44 0.49 0.50 1.32 LHS21 F58G Y104N  

   

  0.56 1.67 LHS22 F58G I111V 0.15 0.19 0.52 1.33 LHS23 F58G A114G  

   

  0.59 1.59 LHS24 F58G N117S 0.09 0.09 0.53 1.47 LHS25 F58G S125A  

   

  0.56 1.56 LHS26 F58G S132N 0.07 0.07 0.49 1.17 LHS27 F58G P239V  

   

  0.52 1.64 LHS28 S78N Y104N 0.37 0.56 0.67 1.71 LHS29 S78N I111V 0.920.92 0.71 1.53 LHS30 S78N A114G 0.74 0.75 0.48 1.49 LHS31 S78N N117S0.83 0.87 0.61 1.51 LHS32 S78N S125A 0.77 0.79 0.89 1.33 LHS33 S78NS132N 0.81 0.76 0.60 1.38 LHS34 S78N P239V 0.40 0.47 0.50 1.66 LHS35Y104N I111V 0.21 0.31 0.64 1.55 LHS36 Y104N A114G  

  0.06 0.59 1.70 LHS37 Y104N N117S 0.08 0.14 0.45 1.72 LHS38 Y104N S125A0.06 0.09 0.64 1.77 LHS39 Y104N S132N 0.98 1.07 0.74 1.26 LHS40 Y104NP239V  

  0.05 0.69 1.82 LHS41 I111V A114G 0.15 0.16 0.43 1.24 LHS42 I111V N117S0.45 0.43 0.51 1.12 LHS43 I111V S125A 0.37 0.47 0.62 1.21 LHS44 I111VS132N 0.53 0.58 0.41 1.00 LHS45 I111V P239V 0.24 0.31 0.40 1.29 LHS46A114G N117S 0.14 0.15 0.48 1.37 LHS47 A114G S125A 0.20 0.29 0.58 1.41LHS48 A114G S132N 0.24 0.25 0.50 1.14 LHS49 A114G P239V  

  0.07 0.48 1.48 LHS50 N117S S125A 0.31 0.39 0.48 1.27 LHS51 N117S S132N0.35 0.35 0.45 1.12 LHS52 N117S P239V 0.15 0.19 0.38 1.32 LHS53 S125AS132N 0.38 0.51 0.47 1.03 LHS54 S125A P239V 0.09 0.12 0.47 1.59 LHS55S132N P239V 0.16 0.22 0.45 1.29 LHS56 S53G F58G S78N 0.72 0.76 0.56 1.48LHS57 S53G F58G Y104N  

   

  0.52 1.78 LHS58 S53G F58G I111V 0.35 0.43 0.38 1.16 LHS59 S53G F58GA114G 0.10 0.14 0.46 1.37 LHS60 S53G F58G N117S 0.22 0.30 0.41 1.19LHS61 S53G F58G S125A 0.18 0.23 0.44 1.46 LHS62 S53G F58G S132N 0.290.32 0.41 1.01 LHS63 S53G F58G P239V 0.06 0.08 0.49 1.54 LHS64 S53G S78NY104N 0.43 0.63 0.76 1.80 LHS65 S53G S78N I111V 0.97 1.01 0.89 1.69LHS66 S53G S78N A114G 0.85 0.89 0.60 1.63 LHS67 S53G S78N N117S 0.940.92 0.69 1.61 LHS68 S53G S78N S125A 0.78 0.82 0.90 1.37 LHS69 S53G S78NS132N 0.96 0.93 0.75 1.48 LHS70 S53G S78N P239V 0.62 0.66 0.44 1.59LHS71 S53G Y104N I111V 0.25 0.41 0.63 1.73 LHS72 S53G Y104N A114G 0.080.09 0.53 1.75 LHS73 S53G Y104N N117S  

  0.12 0.50 1.68 LHS74 S53G Y104N S125A 0.20 0.23 0.50 1.20 LHS75 S53GY104N S132N 0.94 1.00 1.21 1.10 LHS76 S53G Y104N P239V  

   

  0.54 1.63 LHS77 S53G I111V A114G 0.48 0.51 0.47 1.25 LHS78 S53G I111VN117S 0.66 0.74 0.48 1.17 LHS79 S53G I111V S125A 0.49 0.64 0.72 1.27LHS80 S53G I111V S132N 0.78 0.80 0.54 1.19 LHS81 S53G I111V P239V 0.320.38 0.43 1.26 LHS82 S53G A114G N117S 0.16 0.29 0.47 1.19 LHS83 S53GA114G S125A 0.32 0.44 0.50 1.19 LHS84 S53G A114G S132N 0.44 0.50 0.401.05 LHS85 S53G A114G P239V 0.15 0.31 0.44 1.42 LHS86 S53G N117S S125A0.50 0.56 0.54 1.08 LHS87 S53G N117S S132N 0.54 0.59 0.49 1.15 LHS88S53G N117S P239V 0.28 0.41 0.50 1.31 LHS89 S53G S125A S132N 0.50 0.550.67  

  LHS90 S53G S125A P239V 0.06 0.12 0.59  

  LHS91 S53G S132N P239V 0.27 0.33 0.49  

  LHS92 F58G S78N Y104N  

   

  0.67  

  LHS93 F58G S78N I111V 0.17 0.18 0.56  

  LHS94 F58G S78N A114G  

  0.07 0.67  

  LHS95 F58G S78N N117S 0.11 0.12 0.64  

  LHS96 F58G S78N S125A 0.11 0.15 0.65  

  LHS97 F58G S78N S132N 0.06 0.12 0.57  

  LHS98 F58G S78N P239V  

   

  0.60  

  LHS99 F58G Y104N I111V  

   

  0.57  

  LHS100 F58G Y104N A114G  

   

  0.59  

  LHS101 F58G Y104N N117S  

   

  0.61  

  LHS102 F58G Y104N S125A  

   

  0.61  

  LHS103 F58G Y104N S132N 0.19 0.28 0.48  

  LHS104 F58G Y104N P239V 0.05  

  0.62  

  LHS105 F58G I111V A114G  

   

  0.62  

  LHS106 F58G I111V N117S  

   

  0.58  

  LHS107 F58G I111V S125A  

   

  0.53  

  LHS108 F58G I111V S132N  

   

  0.52  

  LHS109 F58G I111V P239V  

   

  0.55  

  LHS110 F58G A114G N117S  

   

  0.64  

  LHS111 F58G A114G S125A  

   

  0.62  

  LHS112 F58G A114G S132N  

   

  0.58  

  LHS113 F58G A114G P239V  

   

  0.45  

  LHS114 F58G N117S S125A  

   

  0.60  

  LHS115 F58G N117S S132N  

   

  0.55  

  LHS116 F58G N117S P239V  

   

  0.52  

  LHS117 F58G S125A S132N  

   

  0.62  

  LHS118 F58G S125A P239V  

   

  0.62  

  LHS119 F58G S132N P239V  

   

  0.65  

  LHS120 S78N Y104N I111V 0.33 0.44 0.63  

  LHS121 S78N Y104N A114G  

   

  0.68  

  LHS122 S78N Y104N N117S  

  0.13 0.60  

  LHS123 S78N Y104N S125A  

  0.06 0.69  

  LHS124 S78N Y104N S132N 1.02 1.00 0.77  

  LHS125 S78N Y104N P239V 0.05  

  0.65  

  LHS126 S78N I111V A114G 0.35 0.36 0.60  

  LHS127 S78N I111V N117S 0.52 0.53 0.54  

  LHS128 S78N I111V S125A 0.51 0.57 0.67  

  LHS129 S78N I111V S132N 0.50 0.46 0.49  

  LHS130 S78N I111V P239V 0.27 0.29 0.55  

  LHS131 S78N A114G N117S 0.11 0.14 0.50  

  LHS132 S78N A114G S125A 0.27 0.27 0.51  

  LHS133 S78N A114G S132N 0.23 0.21 0.56  

  LHS134 S78N A114G P239V 0.12 0.11 0.54  

  LHS135 S78N N117S S125A 0.39 0.48 0.62  

  LHS136 S78N N117S S132N 0.42 0.47 0.55  

  LHS137 S78N N117S P239V 0.09 0.05 0.53  

  LHS138 S78N S125A S132N 0.46 0.47 0.56  

  LHS139 S78N S125A P239V 0.06 0.08 0.64  

  LHS140 S78N S132N P239V 0.21 0.16 0.45  

  LHS141 Y104N I111V A114G  

  0.06 0.63  

  LHS142 Y104N I111V N117S 0.05 0.05 0.57  

  LHS143 Y104N I111V S125A 0.05 0.09 0.66  

  LHS144 Y104N I111V S132N 0.87 0.91 0.60  

  LHS145 Y104N I111V P239V  

   

  0.61  

  LHS146 Y104N A114G N117S  

   

  0.53  

  LHS147 Y104N A114G S125A  

   

  0.50  

  LHS148 Y104N A114G S132N 0.32 0.45 0.50  

  LHS149 Y104N A114G P239V 0.05 0.05 0.62  

  LHS150 Y104N N117S S125A 0.05  

  0.56  

  LHS151 Y104N N117S S132N 0.53 0.63 0.54  

  LHS152 Y104N N117S P239V 0.06  

  0.66  

  LHS153 Y104N S125A S132N 0.30 0.41 0.69  

  LHS154 Y104N S125A P239V  

   

  0.54  

  LHS155 Y104N S132N P239V 0.25 0.36 0.50  

  LHS156 I111V A114G N117S  

  0.05 0.54  

  LHS157 I111V A114G S125A 0.05 0.06 0.59  

  LHS158 I111V A114G S132N  

  0.05 0.58  

  LHS159 I111V A114G P239V  

   

  0.63  

  LHS160 I111V N117S S125A 0.10 0.10 0.66  

  LHS161 I111V N117S S132N  

   

  0.55  

  LHS162 I111V N117S P239V  

   

  0.55  

  LHS163 I111V S125A S132N  

  0.14 0.56  

  LHS164 I111V S125A P239V  

   

  0.60  

  LHS165 I111V S132N P239V  

   

  0.57  

  LHS166 A114G N117S S125A  

   

  0.57  

  LHS167 A114G N117S S132N  

  0.07 0.52  

  LHS168 A114G N117S P239V  

   

  0.67  

  LHS169 A114G S125A S132N  

   

  0.59  

  LHS170 A114G S125A P239V  

   

  0.63  

  LHS171 A114G S132N P239V  

   

  0.55  

  LHS172 N117S S125A S132N  

  0.05 0.56  

  LHS173 N117S S125A P239V  

   

  0.56  

  LHS174 N117S S132N P239V  

   

  0.57  

  LHS175 S125A S132N P239V  

   

  0.55  

  LHS176 N76D D120H K213N M222Q 0.22 0.31 0.68  

 

The LAS/EDTA stability of the BPN′ triple variants was tested andcompared to BPN′-Y217L. The LAS/EDTA assay was performed as described inthe “LAS Stability Assay” section of Example 1, except that the stressbuffer contained 0.1% LAS+10 mM EDTA and the stress plates wereincubated at 35° C. for 25 minutes. The functionality of BPN′ variantswas quantified as a performance index (Pi), which is the ratio ofperformance of a variant to parent protein BPN′-Y217L. Results are shownin Table 4-3.

TABLE 4-3 LAS/EDTA Stability Results for BPN' Variants Pi VariantLAS/EDTA N62Q-G97A-Y217Q 1.26 G97A-N123G-Y217Q 0.95 G97A-G128A-Y217Q1.24 FNA: G97G-G128G- 1.00 Y217L

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. Those of skill in the art readily appreciate thatthe present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those inherenttherein. The compositions and methods described herein arerepresentative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. It is readilyapparent to one skilled in the art that varying substitutions andmodifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by herein.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notexcised material is specifically recited herein.

What is claimed is:
 1. An isolated subtilisin variant comprising atleast one set of the following substitution sets: G97A-G128A-Y217Q,G97A-L126A-G128A, G97A-L126A-G128A-Y217Q, G97A-L126A-Y217Q,G97A-M124V-G128A, G97A-M124V-G128A-Y217Q, G97A-M124V-L126A,G97A-M124V-L126A-G128A, G97A-M124V-L126A-Y217Q, G97A-M124V-Y217Q,G97A-N123G-G128A, G97A-N123G-G128A-Y217Q, G97A-N123G-L126A,G97A-N123G-L126A-G128A, G97A-N123G-L126A-Y217Q, G97A-N123G-M124V,G97A-N123G-M124V-G128A, G97A-N123G-M124V-L126A, G97A-N123G-M124V-Y217Q,G97A-N123G-Y217Q, L126A-G128A-Y217Q, L96T-G128A-Y217Q, L96T-G97A-G128A,L96T-G97A-G128A-Y217Q, L96T-G97A-L126A, L96T-G97A-L126A-G128A,L96T-G97A-L126A-Y217Q, L96T-G97A-M124V, L96T-G97A-M124V-G128A,L96T-G97A-M124V-L126A, L96T-G97A-M124V-Y217Q, L96T-G97A-N123G,L96T-G97A-N123G-G128A, L96T-G97A-N123G-L126A, L96T-G97A-N123G-M124V,L96T-G97A-N123G-Y217Q, L96T-G97A-Y217Q, L96T-L126A-G128A,L96T-L126A-G128A-Y217Q, L96T-L126A-Y217Q, L96T-M124V-G128A,L96T-M124V-G128A-Y217Q, L96T-M124V-L126A, L96T-M124V-L126A-G128A,L96T-M124V-L126A-Y217Q, L96T-M124V-Y217Q, L96T-N123G-G128A,L96T-N123G-G128A-Y217Q, L96T-N123G-L126A, L96T-N123G-L126A-G128A,L96T-N123G-L126A-Y217Q, L96T-N123G-M124V, L96T-N123G-M124V-G128A,L96T-N123G-M124V-L126A, L96T-N123G-M124V-Y217Q, L96T-N123G-Y217Q,M124V-G128A-Y217Q, M124V-L126A-G128A, M124V-L126A-G128A-Y217Q,M124V-L126A-Y217Q, N123G-G128A-Y217Q, N123G-L126A-G128A,N123G-L126A-G128A-Y217Q, N123G-L126A-Y217Q, N123G-M124V-G128A,N123G-M124V-G128A-Y217Q, N123G-M124V-L126A, N123G-M124V-L126A-G128A,N123G-M124V-L126A-Y217Q, N123G-M124V-Y217Q, N62Q-G128A-Y217Q,N62Q-G97A-G128A, N62Q-G97A-G128A-Y217Q, N62Q-G97A-L126A,N62Q-G97A-L126A-G128A, N62Q-G97A-L126A-Y217Q, N62Q-G97A-M124V,N62Q-G97A-M124V-G128A, N62Q-G97A-M124V-L126A, N62Q-G97A-M124V-Y217Q,N62Q-G97A-N123G, N62Q-G97A-N123G-G128A, N62Q-G97A-N123G-L126A,N62Q-G97A-N123G-M124V, N62Q-G97A-N123G-Y217Q, N62Q-G97A-Y217Q,N62Q-L126A-G128A, N62Q-L126A-G128A-Y217Q, N62Q-L126A-Y217Q,N62Q-L96T-G128A, N62Q-L96T-G128A-Y217Q, N62Q-L96T-G97A,N62Q-L96T-G97A-G128A, N62Q-L96T-G97A-L126A, N62Q-L96T-G97A-M124V,N62Q-L96T-G97A-N123G, N62Q-L96T-G97A-Y217Q, N62Q-L96T-L126A,N62Q-L96T-L126A-G128A, N62Q-L96T-L126A-Y217Q, N62Q-L96T-M124V,N62Q-L96T-M124V-G128A, N62Q-L96T-M124V-L126A, N62Q-L96T-M124V-Y217Q,N62Q-L96T-N123G, N62Q-L96T-N123G-G128A, N62Q-L96T-N123G-L126A,N62Q-L96T-N123G-M124V, N62Q-L96T-N123G-Y217Q, N62Q-L96T-Y217Q,N62Q-M124V-G128A, N62Q-M124V-G128A-Y217Q, N62Q-M124V-L126A,N62Q-M124V-L126A-G128A, N62Q-M124V-L126A-Y217Q, N62Q-M124V-Y217Q,N62Q-N123G-G128A, N62Q-N123G-G128A-Y217Q, N62Q-N123G-L126A,N62Q-N123G-L126A-G128A, N62Q-N123G-L126A-Y217Q, N62Q-N123G-M124V,N62Q-N123G-M124V-G128A, N62Q-N123G-M124V-L126A, N62Q-N123G-M124V-Y217Q,and N62Q-N123G-Y217Q, wherein said substitutions are at positionsequivalent to the positions of BPN′ subtilisin set forth in SEQ ID NO:1.2. An isolated subtilisin variant comprising at least one set of thefollowing substitution sets: G97N-G128A-Y217M, G97G-G128S-Y217E,G97A-G128A-Y217Q, G97M-G128S-Y217E, G97A-G128S-Y217Q, G97D-G128S-Y217Q,G97M-G128G-Y217M, G97G-G128S-Y217Q, G97S-G128S-Y217Q, G97G-G128A-Y217Q,G97S-G128A-Y217E, G97A-G128S-Y217L, G97A-G128A-Y217N, G97Q-G128S-Y217L,G97A-G128A-Y217M, G97A-G128A-Y217S, G97D-G128A-Y217Q, G97M-G128S-Y217Q,G97Q-G128G-Y217D-S87Y, G97S-G128A-Y217N, G97A-G128S-Y217T,G97D-G128S-Y217E, G97D-G128A-Y217L, G97G-G128S-Y217E-S78P-A272T,G97T-G128S-Y217D, G97D-G128A-Y217I, G97Q-G128S-Y217Q, G97G-G128A-Y217D,G97Q-G128A-Y217N, G97S-G128A-Y217M, G97S-G128S-Y217N, G97S-G128S-Y217M,G97E-G128S-Y217M, G97S-G128P-Y217Q, G97T-G128S-Y217Q,G97D-G128S-Y217Q-A73T, G97E-G128S-Y217N, G97G-G128A-Y217I,G97Q-G128A-Y217D, G97Q-G128S-Y217M, G97R-G128T-Y217Q-S162P,G97S-G128S-Y217D, G97T-G128P-Y217I, G97Q-G128G-Y217E, G97C-G128G-Y217N,G97D-G128S-Y217H, G97M-G128S-Y217L, G97M-G128S-Y217N, G97S-G128S-Y217E,G97M-G128S-Y217I, G97A-G128P-Y217A, G97R-G128S-Y217D, G97D-G128A-Y217D,G97V-G128G-Y217D, G97V-G128G-Y217E, G97A-G128G-Y217T, G97G-G128N-Y217L,G97D-G128A-Y217T, G97M-G128A-Y217E, and G97M-G128A-Y217N, wherein saidsubstitutions are at positions equivalent to the positions of BPN′subtilisin set forth in SEQ ID NO:1.
 3. An isolated subtilisin variantcomprising at least one set of the following substitution sets:S24R-S87D-Q206E, P40E-A144K-K213L, N61E-P129E-S159K, S87D-S162K-K265N,S87D-T242R-Q275E, N61E-Q103E-N240K, S87D-T242R-K265N, N62R-K265N-Q275E,P129E-S145D-N240K, P129E-P239R-K265N, Q103E-P129E-T242R,P40E-N61E-S87D-S162K-T242R, S24R-N62R-S87D-S145D-K265N,P40E-N62R-S87D-Q103E-S162K, S24R-P40E-S145D-S159K-K213L,S24R-S87D-A144K-K265N-Q275E, N61E-P129E-S162K-K213L-N240K,N61E-S145D-S162K-K213L-T242R, S87D-A144K-S145D-S159K-Q275E,S24R-P129E-Q206E-N240K-K265N, N61E-Q103E-A144K-K213L-T242R,N62R-S159K-Q206E-K265N-Q275E, S24R-Q103E-P129E-N240K-K265N,N61E-Q103E-P129E-P239R-N240K, P129E-S145D-N240K-T242R-K265N,Q103E-S162K-Q206E-K213L-P239R, P40E-N61E-N62R-S87D-S159K-S162K-K265N,S24R-P40E-N61E-A144K-Q206E-K213L-T242R,P40E-N61E-S87D-P129E-S159K-S162K-T242R,P40E-N62R-S87D-S145D-S159K-S162K-Q275E,P40E-N62R-S87D-Q103E-A144K-S159K-Q275E,P40E-N62R-S87D-S159K-S162K-K265N-Q275E,P40E-N61E-P129E-A144K-S162K-K213L-N240K,P40E-N61E-Q103E-A144K-S159K-S162K-Q275E,P40E-N61E-Q103E-S159K-S162K-K213L-P239R,P40E-N61E-Q103E-S159K-S162K-K213L-N240K,N62R-S87D-P129E-S145D-S159K-S162K-Q275E,S24R-N61E-Q103E-P129E-K213L-N240K-T242R,P40E-N62R-S145D-S159K-S162K-Q206E-Q275E,N62R-S87D-S145D-S159K-S162K-K265N-Q275E,N61E-S87D-Q103E-S159K-S162K-K213L-T242R,S24R-N61E-Q103E-P129E-Q206E-P239R-N240K,S24R-N62R-P129E-S145D-P239R-K265N-Q275E,S24R-N62R-P129E-Q206E-N240K-K265N-Q275E,P40E-S145D-S159K-S162K-K213L-P239R-Q275E,N61E-Q103E-A144K-Q206E-K213L-N240K-T242R,S24R-Q103E-P129E-S145D-P239R-N240K-K265N,N61E-Q103E-P129E-K213L-P239R-N240K-T242R,N61E-Q103E-Q206E-K213L-P239R-N240K-T242R,S24R-P40E-N61E-S87D-Q103E-S159K-S162K-K213L-N240K,N61E-N62R-S87D-Q103E-S159K-S162K-K213L-T242R-Q275E,P40E-N62R-S87D-S145D-S159K-S162K-N240K-K265N-Q275E,N62R-S87D-S145D-S159K-S162K-K213L-N240K-K265N-Q275E,S24R-N61E-Q103E-P129E-Q206E-K213L-P239R-N240K-T242R,S24R-N61E-Q103E-P129E-S145D-P239R-N240K-T242R-K265N,N61E-S87D-Q103E-P129E-S159K-S162K-K213L-N240K-T242R,P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,S24R-Q103E-P129E-S145D-Q206E-P239R-N240K-T242R-K265N,N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,S24R-P40E-N61E-S87D-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,S24R-P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R-K265N,S24R-P40E-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,S24R-P40E-N61E-Q103E-P129E-A144K-S145D-K213L-P239R-N240K-T242R,S24R-N61E-S87D-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,P40E-N61E-S87D-Q103E-S145D-S159K-S162K-K213L-P239R-N240K-T242R,S24R-P40E-N61E-Q103E-P129E-S162K-Q206E-K213L-P239R-N240K-T242R,S24R-N61E-Q103E-P129E-A144K-S145D-Q206E-K213L-P239R-N240K-T242R,P40E-N61E-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R,S24R-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R-K265N, andS24R-P40E-N61E-S87D-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R,wherein said substitutions are at positions equivalent to the positionsof BPN′ subtilisin set forth in SEQ ID NO:1.
 4. A subtilisin variantcomprising the substitutions G97A/G128A/Y217Q and further comprising atleast one modification of claim 3, and wherein the positions correspondto the positions of BPN′ subtilisin of SEQ ID NO:1.
 5. An isolatedsubtilisin variant comprising at least one set of the followingsubstitution sets: S53G-F58G, S53G-S78N, S53G-Y104N, S53G-I111V,S53G-A114G, S53G-N117S, S53G-S125A, S53G-S132N, S53G-P239V, F58G-S78N,F58G-Y104N, F58G-I111V, F58G-A114G, F58G-N117S, F58G-S125A, F58G-S132N,F58G-P239V, S78N-Y104N, S78N-I111V, S78N-A114G, S78N-N117S, S78N-S125A,S78N-S132N, S78N-P239V, Y104N-I111V, Y104N-A114G, Y104N-N117S,Y104N-S125A, Y104N-S132N, Y104N-P239V, I111V-A114G, I111V-N117S,I111V-S125A, I111V-S132N, I111V-P239V, A114G-N117S, A114G-S125A,A114G-S132N, A114G-P239V, N117S-S125A, N117S-S132N, N117S-P239V,S125A-S132N, S125A-P239V, S132N-P239V, S53G-F58G-S78N, S53G-F58G-Y104N,S53G-F58G-I111V, S53G-F58G-A114G, S53G-F58G-N117S, S53G-F58G-S125A,S53G-F58G-S132N, S53G-F58G-P239V, S53G-S78N-Y104N, S53G-S78N-I111V,S53G-S78N-A114G, S53G-S78N-N117S, S53G-S78N-S125A, S53G-S78N-S132N,S53G-S78N-P239V, S53G-Y104N-I111V, S53G-Y104N-A114G, S53G-Y104N-N117S,S53G-Y104N-S125A, S53G-Y104N-S132N, S53G-Y104N-P239V, S53G-I111V-A114G,S53G-I111V-N117S, S53G-I111V-S125A, S53G-I111V-S132N, S53G-I111V-P239V,S53G-A114G-N117S, S53G-A114G-S125A, S53G-A114G-S132N, S53G-A114G-P239V,S53G-N117S-S125A, S53G-N117S-S132N, S53G-N117S-P239V, S53G-S125A-S132N,S53G-S125A-P239V, S53G-S132N-P239V, F58G-S78N-Y104N, F58G-S78N-I111V,F58G-S78N-A114G, F58G-S78N-N117S, F58G-S78N-S125A, F58G-S78N-S132N,F58G-S78N-P239V, F58G-Y104N-I111V, F58G-Y104N-A114G, F58G-Y104N-N117S,F58G-Y104N-S125A, F58G-Y104N-S132N, F58G-Y104N-P239V, F58G-I111V-A114G,F58G-I111V-N117S, F58G-I111V-S125A, F58G-I111V-S132N, F58G-I111V-P239V,F58G-A114G-N117S, F58G-A114G-S125A, F58G-A114G-S132N, F58G-A114G-P239V,F58G-N117S-S125A, F58G-N117S-S132N, F58G-N117S-P239V, F58G-S125A-S132N,F58G-S125A-P239V, F58G-S132N-P239V, S78N-Y104N-I111V, S78N-Y104N-A114G,S78N-Y104N-N117S, S78N-Y104N-S125A, S78N-Y104N-S132N, S78N-Y104N-P239V,S78N-I111V-A114G, S78N-I111V-N117S, S78N-I111V-S125A, S78N-I111V-S132N,S78N-I111V-P239V, S78N-A114G-N117S, S78N-A114G-S125A, S78N-A114G-S132N,S78N-A114G-P239V, S78N-N117S-S125A, S78N-N117S-S132N, S78N-N117S-P239V,S78N-S125A-S132N, S78N-S125A-P239V, S78N-S132N-P239V, Y104N-I111V-A114G,Y104N-I111V-N117S, Y104N-I111V-S125A, Y104N-I111V-S132N,Y104N-I111V-P239V, Y104N-A114G-N117S, Y104N-A114G-S125A,Y104N-A114G-S132N, Y104N-A114G-P239V, Y104N-N117S-S125A,Y104N-N117S-S132N, Y104N-N117S-P239V, Y104N-S125A-S132N,Y104N-S125A-P239V, Y104N-S132N-P239V, I111V-A114G-N117S,I111V-A114G-S125A, I111V-A114G-S132N, I111V-A114G-P239V,I111V-N117S-S125A, I111V-N117S-S132N, I111V-N117S-P239V,I111V-S125A-S132N, I111V-S125A-P239V, I111V-S132N-P239V,A114G-N117S-S125A, A114G-N117S-S132N, A114G-N117S-P239V,A114G-S125A-S132N, A114G-S125A-P239V, A114G-S132N-P239V,N117S-S125A-S132N, N117S-S125A-P239V, N117S-S132N-P239V,S125A-S132N-P239V, N76D-D120H-K213N-M222Q, wherein said substitutionsare at positions equivalent to the positions of BPN′ subtilisin setforth in SEQ ID NO:1.
 6. A subtilisin variant comprising thesubstitutions G97A/G128A/Y217Q and further comprising at least onemodification of claim 5, and wherein the positions correspond to thepositions of BPN′ subtilisin of SEQ ID NO:1.
 7. An isolated nucleic acidencoding a subtilisin variant set forth in any of claims 1-6.
 8. Anexpression vector comprising said nucleic acid of claim
 7. 9. A hostcell comprising said expression vector of claim
 6. 10. A cleaningcomposition comprising at least one subtilisin variant set forth in anyof claims 1-7.
 11. The cleaning composition of claim 10, wherein saidcleaning composition is a laundry detergent.
 12. The laundry detergentof claim 11, wherein said laundry detergent is a heavy duty liquidlaundry detergent.
 13. The cleaning composition of claim 10, whereinsaid cleaning composition is a dish detergent.
 14. The cleaningcomposition of any of claims 10-13, further comprising one or moreadditional enzymes or enzyme derivatives selected from the groupconsisting of hemicellulases, peroxidases, proteases, cellulases,xylanases, lipases, phospholipases, esterases, cutinases, pectinases,keratinases, reductases, oxidases, phenol oxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof.
 15. The cleaning composition of claim10, further comprising at least one stabilizing agent.
 16. A cleaningcomposition comprising at least 0.0001 weight percent of at least onesubtilisin variant of any of claims 1-7, and optionally, at least onesuitable adjunct ingredient.
 17. A method of cleaning, said methodcomprising the steps of: a) contacting a surface and/or an articlecomprising a fabric with the cleaning composition of any of claims10-16; and b) optionally washing and/or rinsing said surface or article.18. An animal feed comprising at least one subtilisin variant of any ofclaims 1-7.
 19. A food processing composition comprising at least onesubtilisin variant of any of claims 1-7.